Green fluorescent protein

Green Fluorescent Protein I
This lab is an illustration of protein purification and protein properties. Background I: Green Fluorescent Protein Fluorescence is a process where a molecule absorbs light at one wavelength and then. ... Where is the GFP? Green Fluorescent Protein I - 9. 18) Transfer the column to collection tube 2. Using the rinsed pipette and the same loading technique described above, add 250 µl of Wash Buffer and let the entire volume flow into the column.
Lab 9
What do you predict you’ll see when you compare these two? Explain. 3. Fully denatured GFP does not fluoresce. What critical feature is lost from its structure during this process? ... bacteria with a jellyfish gene and used column. chromatography to purify the Green Fluorescent Protein (GFP). In this lab, we will learn another technique, protein. electrophoresis, which will allow us to visualize both the total. proteins in our transformed E. coli as well as the results of our.
Green Fluorescent Protein (GFP)
In this lab, you will pick one white colony from your LB/amp plate and one green colony from your LB/amp/ara plate for propagation in separate liquid cultures. Since it is hypothe-sized that the cells contain the Green Fluorescent Protein, and it is this protein we want to produce and purify, your first consideration might involve thinking of how to produce a large ... Minimizing the possibility of outside bacterial contam-ination during an experiment through observance of cleanliness and using careful laboratory techniques. See Appendix C. Process of passing an inoculating loop with bacteria on it across an agar plate.
The green fluorescent | GFP AS AN ACTIVE INDICATOR
Green Fluorescent Protein was discovered by Shimomura et al (1) as a compan-ion protein to aequorin, the famous chemiluminescent protein from Aequorea jellysh. In a footnote to their account of aequorin purication, they noted that “a protein giving solutions that look slightly greenish in sunlight through ... (38, 39). However, for other applications it would be desirable to have a GFP that works well at 37?C. The most extensive attempt to develop such a mutant while preserving the complex wild-type spectrum utilized DNA shufing (40), a technique for re-combining various mutations while creating new ones.
Green-Fluorescent Protein Purification Short Course
Using the remarkable Green-Fluorescent Protein (GFP)*, A Novel Marker For Gene Expression, as the source material. ... This feature provides a continuity of purpose, integrating dozens of preparative and analytical protein techniques in a way that few competing courses can match. A problem-solving approach will be used throughout the course. Under the guidance of experienced lab instructors, participants will work in groups of three to plan their own protocols, analyze data, and interpret results.
23 December
Biotechnology Explorer
GFP fluoresces a brilliant green color when viewed using a hand held long wave ultraviolet light (pocket geol-ogy lamp). The gene for the Green Fluorescent Protein was originally isolated from the jellyfish, Aequorea victoria. ... You will have to decide what is best for your students, based upon their lab experience and familiarity with these techniques. Transferring Bacterial Colonies from Agar Plates to Microtubes.
Green Fluorescent Protein Lab Report (24 pt)
Texas A&M University-Corpus Christi CHEM4402 - Biochemistry Laboratory II Green Fluorescent Protein Lab Report (24 pt). 1. Title Page and abstract. ... All figures should be appropriately numbered and have a one-sentence descriptive title. a. Laboratory 7. Figure of GFP cDNA sequence. Be sure that primer binding sites, the Csp45I restriction site and the chromophore are highlighted and described in the figure legend. (3 pt).
Fluorescent Protein Biosensors and Autophagy Laboratory
GFP-like proteins. Green fluorescent protein (GFP) is just one member of a large super-family. ... Advanced fluorescence imaging techniques: lifetime imaging and FRET imaging. Some relevant publications from our laboratory. ‘Ultramarine, a chromoprotein acceptor for Forster resonance energy transfer' Anne Pettikiriarachchi, Lan Gong, Matthew A. Perugini, Rodney J. Devenish and Mark Prescott PloS One (2012) 7 (7):e41028.
4 July
Transformation and Protein Purification of GFP
One of the genes that are. used to visualize fish cells is the Green Fluorescent Protein, or GFP. ... Grade Level 10-­?12th Unit: Genetic Engineering/Recombination Techniques Objectives: 1. SWBAT use manipulatives to plan out the transformation protocol 2. SWBAT use proper lab techniques to transform bacteria with the GFP gene 3. SWBAT use chromatography protocol to isolate and purify the GFP gene 4. SWBAT identify uses of transformation in the biotechnology world Timing: 1st Day: Transformation PowerPoint; Manipulative Exercise & Pour agar plates for day 2 (45 – 90 minutes, depending.
Green Fluorescent Protein - GFP History - Marty Chalfie
As part of the talk, the speaker mentioned GFP. The fact that it was a protein that was inherently fluorescent got Chalfie very excited. He immediately began envisioning ways he would be able to use GFP in his C. elegans research. ... After he published the GFP sequence in Gene, Prasher sent a GFP clone to Chalfie, who gave it to a graduate student, Ghia Euskirchen, who was doing a rotation in his lab. She succeeded in incorporating the GFP gene into E. coli so that they fluoresced green when they were irradiated with blue light!
7 March
COC Biotechnology
Green Fluorescent Protein (GFP). Western Blotting. Macromolecular model of two GFP proteins Version 10-26-05. ... Blotting techniques allow the researcher to transfer protein or nucleic acids to a membrane that is more stable than the gel in which they were initially separated. The transfer technique may use a form of simple capillary action or as in our lab, a blotting device that uses an electrical current to transfer the proteins.
Fluorescent proteins: a cell biologist’s
Fluorescent Proteins (FPs) have revolutionized cell biology. ... For FP-fusion proteins, our lab primarily uses the FPs listed in the rst part of Table 1 ... (1995) Quantitative imaging of green uorescent protein in cultured cells: comparison of microscopic techniques, use...
LAB SESSION 1 | important recombinant proteins
today, the green. fluorescent protein. (GFP). Originally. isolated from the jelly. fish Aequorea. Victoria, GFP is now. used in many. pharmacological. ... Attaching. GFP to other proteins of interests makes it. possible to determine the production or even. uptake of proteins being produced. During this lab session, we will. investigate one of the most. widely used techniques in. protein purification, gel column. chromatography (GCC).
Restriction enzyme digestion lab
Formal Lab Report on Restriction Digestion Analysis. Overview: In today’s lab you will use a technique called restriction enzyme mapping to determine the identity of two different plasmids (small circular pieces of DNA). One of these plasmids contains the coding sequence for the jellyfish gene product, green fluorescent protein (GFP), whereas the other plasmid does not.
Bio314: Advanced Cell
Bio314: Advanced Cell Biology. Purification of GFP. Introduction. In the previous lab, you performed a ... The DNA plasmid also contained the GFP gene that codes for the green fluorescent protein (GFP), which transduces blue light to green fluorescent light, and ... To separate the GFP, several techniques need to be performed such as, centrifugation, the ... If the bacteria have been transformed, they will survive and fluoresce green.
LABORATORY 9
Production of a GFP fusion construct In this lab you will be given three different plasmids encoding GFP fusion. proteins X, Y, and Z. In addition, we will use molecular biological techniques to construct a new plasmid that should express fusion protein U. To produce this plasmid, we will insert a piece of coding sequence from a gene from the nematode C. elegans, which was amplified by PCR from genomic DNA, into an appropriate cloning vector. The amplified coding sequence will be fused in frame to the coding sequence of jellyfish green fluorescent protein (GFP).
Purification
Lab 11: Studying Protein Concentration; GFP Purification, Part 1. Today you will begin the process of purifying the GFP protein from the bacterial cells that you transformed with the pGLO plasmid two weeks ago. You will continue the GFP purification procedure next week in Lab 12. Today’s lab will also teach you how to use the spectrophotometer to determine the concentration of a protein solution using the Bradford assay. ... Before examining the two cultures under UV light, predict with your lab group whether each culture will glow green under UV light.
Transformation Kit | Post Lab Cleanup
In this lab activity, students will attempt to cause a wild type strain of E. coli to incorporate and express a plasmid containing a gene that codes for a green fluorescent protein (GFP). ... 1. Understand the transformation procedure using the heat shock method. 2. Describe the importance of marker or reporter genes in molecular biology experiments. 3. Become familiar with the sterile techniques that are used to handle bacteria, including decontamination when experiment is complete.
Molecular Expressions Microscopy Primer: Specialized...
The most widely used of these fluorescent proteins, the green fluorescent protein (GFP) first isolated from the jellyfish Aequorea victoria, can be attached to virtually any protein of interest and still fold into a fluorescent molecule. ... When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.
10 December
Transformation Lab
The purpose of the pre-laboratory techniques activity is to familiarize students with aseptic techniques, micropipette skills, and streak plating. Practicing these skills and techniques prior to performing the wet lab improves student confidence and reduces error in the lab. ... Green Fluorescent Protein (GFP) The green fluorescent protein (GFP) gene is used because of the bioluminescence (ability of an organism to produce color and light) that is produced when the gene is expressed and energy is transferred to it within its host.
Complete the pre-lab assignment due before the laboratory
I. General Laboratory Skills. Sterile Technique With any type of microbiology technique it is important not to introduce contaminating bacteria into the experiment. Bacteria are ubiquitous and are found on fingertips, bench tops, etc., it is important to avoid contaminating surfaces. ... bla Encodes resistance to ampicillin ori Origin of replication allows plasmid to be copied by bacterial DNA replication enzymes GFP Gene encoding green fluorescent protein araC Encodes repressor to prevent transcription (and expression) of the GFP gene.
Green Fluorescent Protein
Green Fluorescent Protein (GFP): Background on GFP & PGLO. GFP Lab Flow Chart. GFP Bunny!!! Return to Biochemistry Homepage.
16 July
Immuno-detection and Conformational Stability
Immuno-detection and Conformational Stability of Green Fluorescent Protein. In today’s lab we will be attempting to specifically detect GFP (and no other protein) in the crude E. coli lysate sample. ... 2.) GFP-E. coli lysate 3.) Affinity purified GFP. Using the technique of Western blotting (or immunoblotting), our goal is to make only the GFP band in the sample visible. To do this we will be using an antibody, an immune system protein, that is specific for its ability to bind GFP.
pGLO Transformation
pGLO Transformation Lesson 1 – Introduction to Transformation In this lab you will perform a procedure known as a genetic transformation. Remember that a gene is a piece of DNA that provides the instructions for making (coding for) a protein which gives an organism a particular trait. ... The gene codes for a Green Fluorescent Protein that causes the jellyfish to fluoresce and glow in the dark.
DOC
The green fluorescent protein and pGLO plasmid
Sterile technique in transformation experiments. Performing a simple transformation experiment. Activity 2. gene transformation and protein purification. The green fluorescent protein and pGLO plasmid. ... These engineered plasmids are sold and can be used to transform bacteria by industry as well as by students in biology labs. The method of creating recombinant plasmid vectors is described in the first Biotechnology subtopic.
13 January
III. evolution of green fluorescent protein-like...
Basic Applications of Fluorescent Proteins A. Protein labeling B. Photobleaching techniques C. Subcellular localizations D. Promoters tracking E. Timers F. Cell and tissue labeling G. DNA and RNA labeling VII. ... Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol Rev 90: 1103–1163, 2010; doi:10.1152/physrev.00038.2009.—Green uorescent protein (GFP) from the jellysh Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded uorescent labels.
C Ourse I nformation - s pring t erm 2016
Specifically, we will be performing an enhancer trap screen, in which the green fluorescent protein will be used to detect random insertions of a “reporter gene” throughout the genome, and will “report” the activities of nearby gene regulatory sequences. ... 2. Show effort and improvement in your lab techniques so you improve your skills of experimental analysis, experimental design and experimental interpretation (this will impact your performance grade).
Cell Biology Lab Module
Lab 3 Transfecting CHO cells with Green Fluorescent Protein/Tubulin Plasmid Concepts: Importance of GFP in biological studies Use of DNA to manipulate cellular expression Use of transfection reagents Hands on techniques: How to properly handle DNA and transfection reagents Optimal conditions for transfection Transfecting CHO cells grown on 6 well plate Personnel Joanne Manaster?main designer, lecturer and prep person Vitaly Stepensky?.
The Potential for Green Fluorescent Protein as a Screening
A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. ... instruction and advice with lab techniques and procedures • Dr. Williams for introducing me to this program and encouraging me to succeed • Neal Stewart for the loan of the prototype GFP • The horticulture department for funding my education through teaching assistantships • My fellow students who have assisted with the generation of...
Green Fluorescent Protein: a protein composed of 238...
GFP Protein Chromatography from transformed E. Coli. OVERALL TIME ESTIMATE: This series of lessons will be covered over a 7?day period. Day 1 will be the discussion of the “Separation Techniques” Power Point, background information reading on paper chromatography and Pre Lab questions. ... The Post Lab questions will also be completed. For homework, students will read Creatures that Glow, and answer questions based on the reading. Day 4 is devoted to background information on bioluminescence and green fluorescent proteins (GFP).
Day 1, Monday
Common Laboratory Equipment Basic Metric System Laboratory Notebook Procedures Laboratory Techniques. ... ? Lab Exercises #1b: Gel Electrophoresis of Dyes (bb_gelelectrophoresisdyes_09) (45 minutes). ... ? PowerPoint Lecture ? Pre-Laboratory Exercises ? Laboratory #7: E. Coli with Blue and Green. Fluorescent Proteins. Evening.
II. Green fluorescent protein markers
Therefore, we have adapted the jellyfish green fluorescent protein for use as a directly visible gene marker in Arabidopsis, and developed genetic and optical techniques that allow us to visualise and manipulate cells within living plants. ... The bright intrinsic green fluorescence of GFP allows it to be directly visualised in transformed cells, and we wished to use the protein as a simple marker for transformation and misexpression studies.
Ion Exchang Pre-Lab Discu Column chromatography Types and principles Focus on: ion exchange chromatography Ion exchange experiment Sample mixture: GFP Types of Column Ion Exchange Gel F Basis for Ion Exchange Gel F ... Obtain sample mixture of two Green fluorescent protein(GFP): negatively charged (anionic) chromophores (26.9 kDa) 395 nm (major peak) (NOTE: d G 238 amino acids Used t Cytoch Highly conserved heme-protein Associated with inner mit Prepare anion e Your colors wil Overview of your ion Separate GFP from cytochrome c Add 0.01 M KOAc first First remove (elute) a Add.
PPT
Resources for Beta-globin and
A number of different research laboratories use GFP in order to facilitate their studies and to further examine their topic of interest. This website provides a wealth of resources, including a historical perspective, practical uses and one research lab’s applications. ... This site provides information about the green fluorescent protein, with specific reference to structures in the Protein Data Bank.
Green Fluorescent Protein (GFP) causes this marine jellyfish...
The araC operon regulation system which is activated in the presence of the sugar arabinose functions as a molecular turn-on switch for the GFP gene; in the presence of arabinose, pGLO transformed cells will produce (or express) the Green Fluorescent Protein and begin to fluoresce in the ... In a lab environment, bacterial transformation can be achieved in different ways. ? Several transformation methods and techniques have been developed by molecular biologists in the past decades to enforce the uptake of foreign genes located on plasmids and/or suitable expression vectors into E.coli bacteria.
26 December
Tissue Culture Laboratory (BIOE342) - Schedule
SECTION 001 SCHEDULE. Lab Time: Mon 1pm-6pm, Wed 1pm-6pm, Fri one hour during afternoon Lab Sessions: Jan 14, 16, 21, 23, 26, 28, 30, Feb 2, 4, 6, 9, 11, 13, 16, 18 Lecture: Fri 2-2:50 pm, Jan 16, 23, 30, Feb 6, 13. IntroductoryMaterial. Cell Survival and Function in vitro. GFP Transfection Assay. Session 1 (W, 14 Jan). Micropipette. Cell Visualization. Sterile technique. Contamination. pH and color. Session 2 (W, 21 Jan). cell concentration. sterile technique.
15 December
Techniques in Synthesis Laboratory
Cycle 10: Green Project, 3 project reports DUE. Cycle 11: Green Project – Last Day for Lab Work. ... • Carefully read about each project before cycle 2 • Make a plan for which cycles you will work on each project • Most projects can be completed in 1 lab period. (Except Fluorescent Protein project) • It is possible to work on more than one project per cycle. (required for Fluorescent Protein project) • Write your reports as soon as you complete each project.
BioS368 Cell Biology Laboratory at
...uorescence staining/labeling techniques that allow observing organelles, proteins ... Fluorescent Proteins (GFP, RFP, Dendra2, etc.) Expression of green (GFP) and red ... Merged uorescence and DIC image. TA, Chuck Fisher. Cell Bio Lab Members, Fall 2015.
Submitted by
Because these techniques are so new to students, they are often intimidated when faced with common biotechnology skills such as micropipetting, loading gels, or spreading agar plates. Labs done only once do not give students confidence with these skills, nor do they help them understand how the different processes are connected. ... In this lab, students amplify the Green Fluorescent Protein gene(GFP) by the Polymerase Chain Reaction (PCR) using a thermocycler that is leased from the company.
Bioprospecting | Purifying Protein by Design
Specifications Purify Green Fluorescent Protein (GFP) from a bacterial culture lysate and. ... SAFETY CONSIDERATIONS Review general lab safety and safety issues specifically related to the use. of the SDS?PAGE technique. In addition, students will use UV lamps to view Green Fluorescent Protein. Ultraviolet radiation can cause damage to eyes and skin. Short-wave UV is more damaging than long-wave UV light.
Nobel Prize in Chemistry: Green Fluorescent Protein
Ribbon diagram of green fluorescent protein, based on the determined X-ray structure. The 2008 Nobel Prize in Chemistry was awarded in three equal parts for the discovery and development of GFP. ... Fluorescence technology is also used to monitor the distance between two proteins that have been tagged using GFP-family proteins. In the technique known as FRET, the emission spectrum of one fluorophore (the donor) matches the excitation spectrum of the other (the acceptor).
2 March
GFP-Based Biosensors
Green fluorescent protein (GFP) is a 27 kD protein consisting of 238 amino acid ... GFP-Based Biosensors 7. In recent, unpublished work in our lab [30], a ... organisms; GFP expression has minimal effect on the cells and can be monitored noninvasively using techniques such as fluorescence mi? croscopy and fluorescence assisted cell sorting ... GFP fusion proteins (generated by combining the fluorescent protein coding region...
GFP-based live microscopy | The green fluorescent protein
Osamu Shimomura Martin Chalfie. Roger Y. Tsien. "for the discovery and development of the green fluorescent protein, GFP". Fluorescent marker for cellular structures. Day and Davidson, Chem Soc Rev, 2009(38) 2887. Image: Lichtman Lab. GFP-based live microscopy. Ding et al., Develop. Biol.
Research Report
The dual-laser technique is applicable for use on flow cytometers equipped with tun-able multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four flu-orescent gene products within living cells. INTRODUCTION. The cloning of the green fluorescent protein (GFP) gene from the lumines-cent jellyfish Aequorea victoria has led to its widespread use as a marker for gene expression in various biological systems (5–7,22).
Green Fluorescent Proteins: Towards
Yet another widely exploited use of GFP fusion proteins is in protein tracking. For many years a technique called fluorescence recovery after photobleaching (FRAP) has been used to study the mobility and diffusion of fluorescent molecules. ... Reverse Phase Chromatography. When tested in our lab organic extraction methods described by Yakhnin et al.[148] resulted in large losses of protein as a result of precipitation at the phase interface, which formed at each stage of the extraction process. Green or yellow precipitated protein collected from the phase interface at any stage of the extraction...
Green Fluorescent
Green fluorescent protein (GFP) has been proposed as a whole-plant marker for field-level applications (1). The GFP gene was first cloned from jellyfish (Aequorea victoria) in 1992, andhas since been modified for specific applications and transformed into many dif-ferent organisms (2-5). GFP monitoring has the potential to track transgenes under large spatial scales utilizing visual or instrumental detection of the char-acteristic green fluorescence of transgenic material.
Procedures for Managing Government-Furnished
If a lab receives GFP/GFE without any notification of the Research Administration Department or the Equipment Manager, the lab must immediately contact both parties to inform them of the arrival. Note: GFP/GFE can NOT be used for any experimental purposes until the Property Administrator verifies shipping information and photographs & affixes property tags to equipment. MANAGING GFP/GFE It is important to constantly track GFP/GFE especially in regards to location, and Primary User’s contact information.
J. Mark Petrash, Ph.D. | Ophthalmology | University of...
We are using a small fluorescent molecule, called Alexa Fluor350 from Molecular Probes, to tag recombinant protein that has been expressed and purified in our lab. ... We have also used cloning to fuse the alpha A gene to the gene for Green Fluorescent Protein (GFP). We can then use fluorescence to monitor intermolecular interactions. When expressed in bacteria, GFP gives off a strong fluorescent signal that fills the bacterial cell volume.
5 September
The Creating a Transgenic Cricket Expressing GFP in...
Incorporating a DNA microinjection technique adapted from the Noji Lab protocol, University of Tokushima Japan, and the piggyBac transposase system, I will transfect Gryllus bimaculatus eggs with the GFP/target gene vectors and transposase mRNA. ... Theoretically, GFP protein will express in the particular subset cells that can still transcribe the target protein. The GFP expressing cells, when viewed with a fluorescent microscopy, will therefore fluoresce green and display neuronal morphology with high resolution.
Genetic transformation of bacteria with
A successful transformation will result in the expression of the green fluorescent protein (GFP) in the bacteria, causing them to glow bright green under long-wave UV light. ... Transformation of Bacteria with GFP. PROCEDURES. It is important to remember that sterile technique is extremely important for this lab. All the materials provided are sterile and proper handling of these supplies and reagents should result in minimal contamination problems.
2008 Nobel Prize in Chemistry
Aequorea photo-organs fluorescing green [The GFP Site]. Aequorea and purified GFP [Woods Hole MBL]. The gene for GFP was cloned at Woods Hole by Dr. Douglas Prasher in 1992. Martin Chalfie (Columbia University) had learned of Prasher's efforts, and recognized that GFP might make an excellent marker for cells. ... Rainbow of fluorescent proteins [Tsien lab]. Glowing bacteria on culture plate [Tsien lab].
2 April
Fluorescence and Its Applications | Laboratory practices
Concepts: Electromagnetic radiation, absorption spectroscopy, emission spectroscopy, fluorescence, green fluorescent protein, and acidity. Materials provided. ... Figure 2: A typical UV-vis absorption spectrum. Emission spectroscopy and fluorescence Emission spectroscopy is a technique that examines the wavelengths and intensities of light emitted by molecules after they are excited by certain means such as photo exciting.
Guidelines for writing Lab Report #1
BIOL110L Lab Report Module 1 Michael Rexach. Introduction: This section must be 2-3 ... The third paragraph could introduce what is known about your GFP tagged protein, its ... “Here, we characterized the cellular location and biochemical fractionation of a GFP-tagged proteins using a combination of microscopy and cell fractionation techniques.” ... Light and fluorescent microscopy of yeast: Preparation of yeast spheroplasts: Cellular...
2010 Lab 7 - CellBiology
Example of Spinning Disc Confocal image - Labeled Mitochondria Movie (1 frame/2 seconds). The laboratory this week will involve a tutorial on microscopy and fluorescent labeling techniques, as well as a visit to the SOMS confocal facility.
5 August
Male-specific GFP marker strain of Drosophila melanogaster.
Y chromosome carrying green fluorescent protein gene driven by the actin 5C promoter (ActGFP. construct, Reichhart and Ferrandon, 1998). Y chromosome is heterochromatin-rich, and recovery of. ... Technique Notes. 225. Acknowledgment: I thank Celeste Berg for gift of Dp(1;Y)y+ P{ry+11}, Bloomington Stock Center for CyO, P{w+mC=ActGFP}JMR1, Misako Taniguchi and Housei Wada for technical assistance.
General Biology 2 - Analysis of Proteins
3 July
2.1. INTRODUCTION The green fluorescent protein...
I also appreciate Dr. Harry Yamamoto for letting me work in his laboratory for some of my experiments. I also enjoyed working with Dr. David Hieber, a very jovial person and thank him for his ideas. It was also great working with my colleagues Gabriel, Peizhu, Jian and Gwen who made the atmosphere in the lab a pleasant environment to work in. ... 106. REFERENCES Billinton N and Knight AW (2001) Seeing the wood through the trees: A review of. Techniques distinguishing green fluorescent protein from endogenous autofluoresence. Analy.
Biology 6B | 12 No Monday Lab 18.5 pGLO 7: Plasmid gel
The laboratory procedures used will emphasize the modern tools and techniques of molecular biology that are used to study cell biology, as well as many other aspects of life sciences. The course lab exercises are organized around five lab project reports. Each report will be a group project and cover experiments conducted over several lab periods. ... In this lab, you’ll use a plasmid to insert a gene for “green fluorescent protein” into E. coli cells. If it works, you’ll get fluorescent bacteria! By the time you finish this lab, you should understand
Green Fluorescent Protein Is Lighting Up Fungal Biology
DigitalCommons@University of Nebraska - Lincoln. Green Fluorescent Protein Is ... Recreating the complex environments in which fungi naturally occur, in a lab or a ... Techniques for eliminating selectable markers from transformed organisms have been...
Proteins as Calcium Sensors and the Effect of
4.1.2. Purification techniques designed for red fluorescent proteins. Red fluorescent proteins like mCherry which emit fluorescence in the 610nm wavelength region in the visible spectrum have been purified using different methods. One common technique is by utilizing their inherent copper binding property. ... Dr. Jenny Yang’s lab has created two GFP calcium sensors from the Green Fluorescent Protein (GFP): EGFP-7E15 or D11and EGFP 172EF-III or G1 (Figure 5.6).
Development and Use of Fluorescent Protein
Fluorescent proteins such as green fluores-cent protein (GFP) from the jellyfish Ae-quorea victoria (Fig. ... Kinetic Microscopy Using Fluorescent Protein Chimeras. New and updated fluorescence imaging methods have been essential to studies exam-ining the localization and kinetic behavior of GFP-tagged proteins. Notable among these techniques are 4D microscopy, fluorescence recovery after photobleaching (FRAP), fluo-rescence loss in photobleaching (FLIP), and fluorescence correlation spectroscopy (FCS).
BioMicroSystems: Labs and
BioMicroSystems: Labs and Fabs for Nanotechnology and Nanomanufacturing. ... GFP Assembly on Chitosan. Green Fluorescent Protein (GFP). ... Techniques to measure response of cells to stimulants.
Jungers Center - Unni Lab | Jungers Center | OHSU
Our recent data demonstrate that we can visualize human wild-type alpha-synuclein fused to enhanced Green Fluorescent Protein (Syn-GFP) in the cortex of transgenic mice with cellular and synaptic resolution using this cranial window-based approach. ... Working as a Senior Research Assistant in Vivek’s lab combines two of my favorite things in science, innovative research techniques and in vivo fluorescence microscopy.
10 November
Imaging > Khokha Lab | Pediatrics | Yale School of Medicine
Unknown. Lab Techniques. ... Ciliary IFT in WT embryo. intraflagellar transport - anterograde and retrograde movement in Xenopus cilia. Xenopus animal caps mem-RFP, H2B-GFP Imaged on Nikon A1R - Robert Huebner Cold Spring Harbor Lab - Xenopus Course. Cell Division.
4 July
Counting of Total Fluorescence in a Cell (Theory) : Biological...
The use of specifically labelled molecules such as genetically encoded fluorescent proteins helps to transform the fluorescent microscopy techniques from a simple localization test to quantitative data for further functional analysis of the cell. Image analysis software thus becomes an important tool for most biologists to evaluate the microscopic fluorescent images into more reliable quantitative manner. In this lab, image analysis techniques are applied to count the total fluorescence in a stained cell using software ImageJ...
24 April
High schoolers use cutting-edge technique to create proteins...
High school students use cutting-edge technique to create fluorescent proteins, thanks to Stanford researchers. ... Many of the students had never been on campus, and cited this part of the experience as a highlight. "It was fascinating to see a real setting of a college lecture," said Nicole Snider, one of the students in the class. The goal of the students' experiment was to create green fluorescent protein (GFP) outside of a living organism.
3 December
Hinkle Lab - Labs - University of Rochester Medical Center
For example, we can "tag" signaling proteins with green fluorescent protein (GFP) and then follow their movements in the cell during hormonal stimulation in real time. We monitor intracellular calcium in the cytoplasm and the endoplasmic reticulum using several fluorescence imaging techniques. In addition, we use biochemical and genetic approaches to identify proteins that interact with the receptor during biosynthesis, signaling and desensitization.
9 July
LAB EXERCISE: Genetic Transformation
In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. ... You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark.
Green Fluorescent Protein (PDB: 4EUL) from Aequorea...
Wild-type green fluorescent protein, or wt-GFP, occurs naturally in the jellyfish Aequorea victoria. wt-GFP has a minor excitation peak at 475 nm and a major excitation peak at 395 nm. ... The major significance of wt-GFP is due to its wide range of applications in the fields of biochemistry and cell biology. wt-GFP has the unique ability to fluoresce without the need for any co-factors or substrates except molecular oxygen. Therefore, wt-GFP can easily be used to tag proteins within a cell and monitor their expressions levels or localization patterns (3). If the gene encoding for wt-GFP is connected in frame with the...
3 June
HetR-gfp
To visualize the pattern of differentiation prior to the appearance of visible heterocysts, the gene encoding green fluorescent protein (from a jellyfish) was fused to the differentiation-specific gene, hetR. This procedure allows hetR expression to be seen as green fluorescence. Hours after inducing differentiation, hetR is expressed in groups of adjacent cells that otherwise appear no different from vegetative cells.
26 October
Biotechnologist for a Week » Boston University Medical Campus
In the purified protein session, students are asked to imagine that they work for a biotechnology company that needs to produce and market green fluorescent protein (GFP). ... To work in the lab, students don white lab coats, blue latex gloves, and plastic safety glasses. At SummerLab, the “biotechnologists” divide into three groups, and each group learns a technique, such as bacterial transformation, cell lysis, or protein purification.
7 August
Lab Review 2
You can count on needing to know the major points for the lab work & tests. ... Know some basic sterile techniques used in microbiology. ... The GFP gene is the Green Fluorescent Protein from the jellyfish Aequorea victoria that fluoresces brilliantly when viewed under...
26 November
Lab 1A: Microscopy I
in which the gene encoding the protein of interest is fused to a gene for the naturally fluorescing Green. Fluorescent Protein (GFP) from the jellyfish Aequorea victoria. ... 5. What advantage did each of these methods give over the Iodine staining used to observe the squamous cheek cells in Lab 1A? (#52). 6. What specialized reagent would need to be made before you could do live imaging of a GFP-tagged protein? (#53).
University of california
The Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria, is commonly used in the biological sciences as a tag on cells and molecules because it. ... Figure 15. Polarization of GFP using one lock-in amplifier at 5 K. To gain sensitivity a cascade lock-in amplifier technique was implemented. The signal is modulated at two different frequencies. The PEM modulates the signal at 40 kHz and an optical chopper modulates at 990Hz.
Fluorescence resonance energy transfer between...
0378-1119/96/$15.00. GENE 09471. Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein *. (FRET; Aequorea victoria;fluorescent proteins; factor Xa). ... (c) Conclusions (1) We have shown that FRET occurs between two. variants of GFP that have been linked together using molecular genetic techniques. Future spectroscopic studies the fusion protein will be directed at determining. more details of this physiochemical process, possibly by the use of time-resolved spectroscopy.
Regeneration | Birnbaum Lab
The root grows continuously by the replenishment of cells within and around the Quiescent Center (QC), which is marked by the green fluorescent protein on the left. ... We have a high-resolution regeneration imaging system in which we can track regenerating roots at cellular resolution for several days. We have also developed single-cell RNA-Seq techniques for single regenerating cells that permit us to ask about their molecular trajectory during regeneration.
11 January
University of Pennsylvania || Gene Therapy Program
Gene Therapy Program Suite 2000 Translational Research Labs (TRL) 125 S. 31st ... 3 ways to detect beta-galactosidase. Techniques. Reporter gene detection. ... Double immunofluorescence labels the macrophage in red with antibodies against Mac-2 and viral protein in green with antibodies against spike. * ... Fluorescent in situ hybridization (FISH) was used to detect replicating DNA (yellow) of an adenoviral vector in 293 cells.
2 May
To fluoresce green
To facilitate microscopic visualization of the location and movement of Verticillium dahliae in mint, a green fluorescing protein (gfp) gene from a jellyfish was inserted into a mint isolate of V ... At present, all transformants are confined by federal regulatory guidelines to controlled conditions in labs, growth ... Previous special staining techniques were much more cumbersome and much thinner ... 1998. Green fluorescent protein expressed by.
Green Fluorescent Protein
• Background Theory of Fluorescence and Fluorescence Resonance Energy Transfer (FRET). • Instrumentation • Application to the study of protein-protein interactions • Comparison to X-Ray Diffraction and Nuclear. Overhauser Effect-NMR • Advantages and Disadvantages • Summary. ... – Non-Specific Labeling – Microinjection into cells. • New Methods and Fluorophores Needed. Green Fluorescent Protein.
UW – Green Bay Research Council
I therefore propose testing the feasibility to perform original experimental research in one of my laboratory courses, Biology 408 – Molecular Biology Lab. ... I plan to have students of the Molecular Biology lab perform this analysis for the rKr2 protein in the fall of this year. ... Beyond training them in experimental techniques, this second approach will give ... Cloning vectors containing the green fluorescent protein are available from my...
FRET Basics and Applications
FRET and Fluorescent Proteins (FPs). ... Comparison of CFP and GFP2 in the same construct. Before. After. CFP-YFP: FRET efficiency 20% GFP2-YFP: FRET efficiency 30% => 50% increase. S. Terjung + T. Zimmermann EAMNET FRET teaching module. Improved FRET Efficiency significantly improves Detection.
Strain for the Use of Green Fluorescent Protein
Also in order to make green fluorescent protein more useful, much of the research being conducted in flies with it utilizes the GAL4-UAS enhancer trap technique, which helps target the expression of green fluorescent protein (Brand and Perrimon, 1993). ... I would also like to thank G. Lee for her tremendous help and input on microinjections, as well as my other fellow lab members for their help and support on this endeavor. 16. Drosophila CRZ-GFP.
BIOL 230 Lab Manual, Lab 17
This is because proteins are usually hundreds of amino acids long and are composed of 20 different amino acids. ... For example, in the direct fluorescent antibody test for Neisseria gonorrhoeae, mentioned briefly in lab 16, the unknown antigen, suspected ... The indirect fluorescent antibody technique involves three different reagents ... When viewed with a fluorescent microscope, the infected cells will fluoresce green.
23 October
croppedCLN2-GFP
The G1/S-regulated promoter for the CLN2 G1 cyclin drives unstable GFP. Gene expression (pulse of green fluorescence) is detectable in every cell cycle, starting around the time of budding.
26 April
2.2 Green Fluorescent Protein (GFP)
1994). GFP was successfully cloned and expressed the protein in Escherichia coli (Figure 2.2) and Caenorhabditis elegans in Martin Chalfie’s lab (Tsien, 1998). These bacteria expressed the green fluorescent when it was induced with isopropyl-?-thiogalactoside (IPTG) (Chalfie et al., 1994). ... A chimera was the resultant of a fusion of cloned gene and GFP using standard subcloning techniques (Zimmer, 2002). GFP fusion tags were used to visualise dynamic cellular events and to monitor the protein localization (Tsien, 1998).
Han lab | creol | Fluorescent Probes
Spinach RNA mimic of green fluorescent protein (GFP): RNA plays essential roles in biology; it transmits genetic information from DNA to proteins and also controls gene expression in various ways. Particularly, its location and copy number strongly affect the development of organisms. ... We are studying important biomarkers of neurodegenerative diseases using human brain samples from Parkinson's and Altzheimer's diseases patients. Advanced bioimaging techniques.
7 March
Fluorescence resonance energy transfer (FRET)
FRET techniques and fluorescent reagents such as GFP, which are widely used to probe for the presence and activity of genes, cellular components, and metabolic or sig-naling pathways, can become more powerful and flexible when multispectral imaging tools are employed. ... 1996. Engineering green fluorescent protein for im-proved brightness, longer wavelengths and fluorescence resonance energy transfer.
RIBS Daily Schedule
M · Introduction/overview of RIBS. · LAB BASICS: Pipetting tutorial (p. 1-7), practice loading gels (p ... W · MICROBIOLOGY BASICS: (sterile technique, streaking) Streaking (p. 5-6). ... GFP. · FLUORESCENT MICROSCOPY live cell stain organelle stains.
The Tg(ccnb1:mcherry) transgenic zebrafish line: using...
Moreover, many transgenic lines are made with GFP reporters, so the unique fluorescence of mCherry is convenient for crossing with GFP reporter lines. Thus far, the Tg(ccnb1:mcherry) line has been crossed to the Tg(gfap:gfp) transgenic line (mi2002 allele name), in which all muller glial cells are labeled with the GFP reporter, which is driven by glial fibrillary acidic protein (GFAP) regulatory elements (Bernardos, 2006). ... According to observations made by the Hyde lab, both 3dpf and 4dpf embryos should give a clear and strong co-localization between PCNA and fluorescent protein in the CMZ.
Green Fluorescent Protein (GFP)
The following stains offer a glimpse of common staining techniques used for cell enumeration and viability; this list is not exhaustive. ... This type of staining involves the insertion of a gene encoding protein, a green fluorescent protein (GFP), into the genome of a bacterium via genetic engineering. When viewed with ultraviolet microscopy, GFP-tagged cells fluoresce green. GFP cannot be used in studying natural populations, as the GFP must be inserted into the genome.
1 October
Retrospective Theses and Dissertations
By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. ... Novel fluorescence imaging techniques have been used to examine localization and kinetic behavior of fluorescent proteins.51,52 These techniques were influenced by developments in both time-lapse microscopy and fluorescent protein probes.
Identifying proteins required for
To identify these proteins, a Green Fluorescent Protein (GFP) protein-trap screen using Drosophila melanogaster was employed. Flies obtained from a GFP protein-trap database known as Flytrap were dissected and the location of the GFP was classified using fluorescent microscopy. ... In the past, lines have been analyzed by other labs, and protein expression was minimally affected by the presence of GFP within its sequence (Morin 2001).
Gfp final reading moodle examination fall 2015
End term examination gfp full time level 1, 2, 3. Day date level. ... General Lab General Lab. Gfp final reading moodle examination fall 2015.
Green Fluorescent Protein Expression in Bone
To address this question in vitro, this study evaluated expression of the reporter gene product green fluorescent protein (GFP) in bone marrow-derived mesenchymal stem cells (BMDMSC) from immunocompetent and athymic nude rats following GFP gene delivery using a first-generation adenoviral vector (Ad).
Gene Mutation to Protein Characterization
Keywords: Enhanced green uorescent protein (EGFP), mutagenesis, protein ... biology, it can be challenging for students to conceptually connect what they have learned from one lab to the next. ... Although there are obvious benets associated with teaching students classic techniques in DNA manipula-tion and protein purication, we chose ... yet many of these were similar to previously published mutations with Green Fluorescent Protein [12...
Here are your gels from the GFP lab
Below are links to your gels. For each lane, measure the bands and put them in table 4. You will use your Lambda HinDIII lane to create a standard curve. See the "DNA" lab if you forget how. Get a "power" trendline for your curve and plug in bands from the other lanes into the formula. If your gel did not turn out well, feel free to use another group's gel for the analysis section. This lab is due Monday, 3/13/17. You can turn this lab individually or as a group.
4 March
Engineering GFP-Family Proteins to Achieve Novel Functions...
ii. Abstract. The Green Fluorescent Protein (GFP) has single handedly revolutionized microscopy and molecular biology. A simple search for GFP in the NCBI Pubmed database yields over 29,900 unique publications as of April 2015 demonstrating the importance and influence GFP has had on the scientific community. ... The Karanicolas lab sought to apply this Trp to Gly mutation followed by rescue by indole to a GFP protein. There is a conserved Trp present on many GFP family proteins located on the central helix of the protein.
Development and Use of Fluorescent Protein Markers in Living
Fluorescent proteins such as green fluores-cent protein (GFP) from the jellyfish Ae-quorea victoria (Fig. ... Kinetic Microscopy Using Fluorescent Protein Chimeras. New and updated fluorescence imaging methods have been essential to studies exam-ining the localization and kinetic behavior of GFP-tagged proteins. Notable among these techniques are 4D microscopy, fluorescence recovery after photobleaching (FRAP), fluo-rescence loss in photobleaching (FLIP), and fluorescence correlation spectroscopy (FCS).
BMC Microbiology
One of the best examples of a technique that has facilitated cell-based studies is marker systems that use fluorescent pro-teins (FPs). Since the green fluorescent protein (GFP) was first discovered from the jellyfish Aequorea victoria in 1962 [5], various types of FPs, including red, yellow, and cya-nine fluorescent ... To get thin transverse sections (0.1 mm), TP309 and TP309-XA21 leaves inoculated with PXO99GFP were cut using a razor blade. The small pieces of leaf sections were placed on a microscope slide, submerged in immersion oil (Cargille lab, USA), covered with a glass slip and sealed with grease.
And sorting by copas ™ flow cytometry technique
The Sxl PE is active only in fly embryos that have a 1:1 ratio between X-chromosomes and autosomes. Consequently, female embryos containing the Sxl PE/EGFP fusion construct exhibit a green fluorescence, while male embryos do not. ... The separate samples were then processed for RT-PCR with sample RNA, as well as, Western blot analysis of protein samples. Figure 1. Dechorionated embryos from G78b (3rd chromosome) viewed by fluorescent microscopy.
Cytochrome C-GFP Reporter Cell Line to Study
figure 3, requested from Douglas R. Green Lab, which contained cytochrome C gene in-frame fused with green fluorescent protein (GFP) gene [25]. ... In this present study, we used molecular biology techniques coupled with time lapse fluorescence microscopy to investigate the release kinetics of cytochrome C-GFP in response to three different apoptosis-inducing reagents; TRAIL, Staurosporine, and Tarceva.
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3 April
Computationally Guided Monomerization of Red
Thank you Niles Pierce for helping me fall in love with science all those years ago as a SURF student in your lab. I enjoyed engineering a control system for a DNA enzyme, and. iv. learned some valuable laboratory techniques and the lay of the land here at Caltech. ... 1.1 Background. Fluorescent protein (FP) engineering emerged as a field in the late 1990’s, shortly after green fluorescent protein (GFP) from Aequorea victoria was first isolated and characterized (1-3).
Impairment of Protein Trafficking upon Overexpression
Reduction of Tf uptake by expression of wild type optineurin-green fluorescence protein (OPTN. ... After 15 min, intense accumulation of red-fluorescent TR-Tf in perinuclear areas were seen in nontransfected (non-green) and GFP mock-transfected green cells (Fig. ... Using siRNA techniques, a previous study [10] showed that downregulation of ... Immunofluorescence microscopy. Cells (18,000 cells/well) on Lab-Tek 8-well CC2 glass...
NANO ENGINEERING
For those that downloaded the notes – a reward! Paste the...
In this experiment, the pGLO plasmid carries the gene (GFP) which produces the green fluorescent protein and a gene (bid) that codes for a protein that gives the bacteria resistance to an antibiotic. ... Techniques Sterile Technique • When working with the inoculation loops, pipetters, and agar plates, the round circle at the end of the loop, the tip of the pipetter, and the surface of the agar plate should not be touched or placed onto contaminating surfaces. • Volume measurement with a graduated dropper pipette.
Fluorescence-guided Single Sensillum Recording | Potter Lab
Fluorescence-guided Single Sensillum Recordings (FgSSR) is a method for performing GFP-targeted SSR in Drosophila. Full details can be found in Lin and Potter, PLoS One, 2015. Traditional single sensillum recordings (SSR) rely on the sizes, shapes, locations and neuronal odor responses (typically to a panel of up to 10 standard odors) to correctly identify target sensilla. ... Homozygous. reporter lines are especially useful when targeting trichoid sensillum. This technique helped clarify the classifcation of intermediate and trichoid sensilla.
11 August
Application of the Split GFP system to Listeria
Generally, proteins are genetically modified so that they are expressed as a fusion to a fluorescent protein, such as Green Fluorescent Protein (GFP). These proteins can then be observed using fluorescence microscopes in real time, where their localizations and dynamics can be tracked relative to other identifiable parts of the cell. Examples of cellular processes that have been studied using fluorescence techniques include the relative time of transcription and localizations before and after protein interaction of interest.4.
Visualization of Cell | Using Fluorescence Microscopy
...pH using fluorescence microscopy involving a ratiometric green fluorescent protein (GFP) ... pH within individual cells in the biofilm fluorescence microscopy techniques. ... throughout this project; I would also like to thank Keith Martinez and the Slonczewski lab.
Center for Plant Cell Biology: REU 2003
When GFP105, a gene that encodes proteins that allow green fluorescence to illuminate, is over expressed by infiltration into a GFP transgenic plant, gene silencing is triggered under normal conditions. Degradation of GFP RNA is thought to occur as indicated by a reddish hue under fluorescent lighting. ... In Dr. Yang's lab, we have been studying the role of Rop (RHO-like GTPase of plants) in cellular signaling. Rop is a small G-protein that acts as a molecular switch in many plant signal transduction pathways. We have been using the emerging technique of chemical genetics in order to discover previously...
17 June
Research Areas - Cedars-Sinai
Therefore, the Jones Lab is using a mouse that has green fluorescent protein only in the cells that regulate pulmonary blood flow (vascular smooth muscle cells) to understand how IL-1b may be playing a role in this disease. The Jones Laboratory is also using novel in vivo imaging techniques to try to determine the functional changes that occur during the development of acute lung injury.
24 November
Additional Projects | Kaverina Lab
We are now investigating the role of MT-binding proteins in stress fiber organization and contractile properties of ... The goal of this project is to utilize a variety of high-resolution live-cell imaging techniques to examine the effect that RASSF1A has on MT ... This project is in collaboration with Geoff Clark's lab at the University of Louisville . ... GFP-Src is shown in green and the MT network is highlighted by mCherry-EMTB, which is shown in red.
28 February
BioE 498 Lab: In-Fusion BioBrick Assembly and Re-engineering
Half of the lab teams will construct a green fluorescent protein (GFP) circuit and the other half will construct a red fluorescent protein (RFP) circuit. ... B0032 is a medium-strength RBS that allows for translation of the GFP or RFP coding sequence into protein. This RBS has a measured activity of 0.3 relative to a stronger RBS B0034. E0040 and E1010 are the GFP and RFP coding sequences, respectively. E0040 comes from jellyfish and there is detectable green fluorescence within 8 minutes after being expressed due to its fast folding time.
Green Fluorescent Protein in Inertially Injected Aqueous...
Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. ... Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.
Department of Cellular and Integrative Physiology
3. Protein-Protein Interactions a. Display potential protein-protein interactions using co-labeling confocal imaging techniques. b. Identify direct protein-protein interactions using co-immunoprecipitation methods. c. Determine globular actin polymerization/depolymerization using biochemical and microscopy techniques. ... ) changes in the cell or isolated membrane using immunofluorescence and enhanced green fluorescent protein tags displaying high/specific affinity for PIP.
Agonist and antagonist activity in a gfp yeast based estrogen
Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence polarization assays have previously been performed using ER to study ligand binding afnities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene ac-tivation. ... Recombinant yeast cells that express ER? use the green uorescent protein (GFP) reporter to determine whether ER?, in the presence of a particular ligand, has activated gene of expression.
Optically modulated fluorescent proteins
Wild type green fluorescent protein Yellow fluorescent protein. xxiii. SUMMARY. ... Combining the SAFIRe technique with the biocompatibility of fluorescent proteins ... Our lab has shown the advantage of modulation in FRET systems previously,207 this could...
McNair Scholars Journal
A fusion between pHdc and the enhanced Green Fluorescent Protein (eGFP) has been made in a plasmid that will allow generation of transformant flies. ... Standard molecular techniques: Techniques used to characterize DNA and clone into bacterial plasmids fol-lowed established protocols (Sambrook et al., 1989). Enzymes were obtained from either Promega Corporation (Madison, WI) or New England Biolabs (Valencia, CA).
Real time PCR | Lab 9 Protein purification
2. Hybridization or FRET probes (Light Cycler) 3. DNA-binding agents (SYBR Green, Eva Green, LC GReen) non-specific fluorescent dyes that intercalate with any double-stranded DNA . Principles of Real-Time Quantitative PCR Techniques (a) SYBR Green I technique ... Technique Of DNA Fingerprinting: LAb 8 Protein separation and quantitation SDS gel : The system most people use for separating proteins by gel electrophoresis was formulated by Laemmli (Nature 227:680-685 [1970]). It is such a commonly used laboratory technique.
Xu lab protocol and instructions
125 ul, 1xPBS, 45 ml]: incubate sections for 1-2 hours in room temperature (0.4 ml per well) l incubate sections with the primary antibodies [1:500 Chicken ?-GFP (Aves Lab) and an antibody for one content] for 4 hours at a shaker and then moved into the 4 deg fridge for 24-36 hours at 4 ?C. ... cover-slip with Vectashield l or dry overnight, then proceed with dehydration and defatting. l keep slices in Xylenes until being cover-slipped with Krystalon in a chemical hood. l examine sections with fluorescent scopes or confocal scope.
Mutations that suppress the thermosensitivity of green...
not necessarily straight forward [4]. for invasive techniques or the addition of cofactors [6,7]. For example, safe expression of high levels of GFP in 1654 Current Biology 1996, Vol 6 No 12 or Vol 7 No 1 transgenic Arabidopsis thaliana has required the elimination Figure 1 of a cryptic intron from ... Transiently in living cells. Curr Biol 1995, 5:635–642. transfected cells were grown on Lab-Tek Chambered Coverglass (Nunc) 15. Kaether C, Gerdes HH: Visualization of protein transport along the for 24 h at 37 °C prior to fluorescence microscopy. For flow cytometric secretory pathway using green fluorescent protein.
17 February
Chick transfection with GFP
2. Zernicka-Goetz, M., Pines, J. (1998) Applications of Green Flourescent Protein in, Tuan, S., R., Lo, C., W., eds. (2000) Developmental Biology Protocols: Method in molecular biology. Human Press: New Jersey.
22 February
Determination of Quinine in Tonic Water by Fluorescence
PRE-LAB ASSIGNMENT. 1. You have the fluorescence emission spectrum of quinine from your first two labs. For maximizing sensitivity and restricting the signal to just quinine, choose the excitation ?ex and emission ?em wavelengths you should use. Explain how you arrived at these values. ... How can we investigate protein-ligand interactions using fluorescence? • Uses of FRET. • Use of GFP (or other fluorescent proteins such as yellow FP, red FP, blue FP) containing specialized protein construct (by constructing the plasmid coding for it) as a generalized protein immobilization kit.
The Lab of Dr. Sharona Gordon
Our lab lab studies the molecular mechanisms of activation and regulation of TRPV1 channels. ... (A) A plasma membrane sheet loaded with fluorescent Rhodamine B C-18. ... I’m interested in membrane protein structure-function with an emphasis on the 6-TM voltage gated ion channel superfamily. I use a broad array of biochemical and biophysical techniques to study the energetics of channel activation/inactivation/closing...
13 December
Green Fluorescent Protein (GFP)
Green fluorescent protein (GFP), from Aequorea coerulescens, has been expressed as a His-tag fusion protein in E. coli cells and purified using metal chelate affinity and gel filtration chromatography. The protein fluoresces at 510 nm due to its natural chromophore that is spontaneously formed after properly folding and remains stable and intact after protein unfolding.
Biotechnology
In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. ... Gene Regulation. Green Fluorescent Protein. Plasmid pGLO Recombinant DNA Technology Screening Sterile Technique Streaking Vector.
Development of an in-cell forster resonance energy
The Green Fluorescent Protein was first discovered in 1962 alongside aequorin, a chemiluminescent protein from Aequorea jellyfish [28]. Since then, it has been studied in numerous biochemical ways including crystallization and the phenomenon of energy transfer. ... In order for atomic-resolution information to be gained from inside living cells and have the technology be easily accessible to the average structural biology lab, we need to utilize a few unrelated techniques. First, bacteria could be used to produce large quantities of recombinant protein.
Movement of yeast cortical actin cytoskeleton
ABSTRACT Fusion proteins between the green fluores-cent protein (GFP) and the cytoskeleton proteins Actlp (actin), Sac6p (yeast fimbrin homolog), and Abplp in ... purchased. from New England Biolabs. Standard techniques and conditions were used. ... Abbreviations: GFP, green fluorescent protein; FITC, fluorescein isothiocyanate. 3886. ... 3 and 4, are available. from the World Wide Web Botstein Lab site at Stanford.
Monitoring Protein-Protein Interactions
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria has exceptional physical and chemical properties besides its spontaneous fluorescence. These properties, which include high thermal stability and resistance to detergents, organic solvents, and pro-teases, endow GFP with enormous potential for biotechnical applications.
Whole 2 body Fluorescent
( green fluorescence protein , GFP). : ( bioluminescence reaction ). ... Fig11 Stable high2level expression GFP transfectants in vitro. (a) fluorescent microscope (200 ) ; (b) laser scanning confocal microscope. 212. ,3 3 . () 213 GFP. 15 mm
Use of Green Fluorescent Protein to Monitor
Digital Commons @ Kent State University Libraries. Use of Green Fluorescent Protein to Monitor Survival of Genetically Engineered Bacteria in Aquatic Environments. ... Many methods for detecting model genetically engineered microorganisms (GEMs) in experimental ecosys-tems rely on cultivation of introduced cells. In this study, survival of Escherichia coli was monitored with the green uorescent protein (GFP) gene.
Dendra2 and PS-CFP2 to track protein movement
Photoactivatable fluorescent proteins (PAFPs) are GFP-like proteins capable of pronounced changes in fluorescence characteristics in response to relatively intense irradiation at a specific wavelength. Today they are becoming widely popular as unique tools allowing precise photolabeling and tracking of mov-ing objects in living systems1. They also open novel perspectives for high resolution imaging techniques2, 3. A whole palette of these proteins is available with va-rious spectral characteristics, photobehaviour and biochemical properties (Table 1).
Laboratory techniques and molecular biology
Lab.and Aseptic Conditions and Basic Stem Cell Laboratory Techniques: Primary, suspension, monolayer and 3D cultures : Isolation of stem cells from bone marrow and adipose tissue for clinical applications. ... 14.00-17.00 : APPLICATION (KOGEM/MOLECULAR BIOLOGY LAB I and II). Protein isolation. SDS-PAGE and Western blotting (M.KASAP, G.DURUKSU, G.AKPINAR, Z.S.UNAL, O.SAGLAM,) Introduction of proteomix lab.
Jim Remington | Green Fluorescent Protein
Email Office: Willamette Hall Room 377 Office Phone: 541-346-5190 Lab: Willamette Hall Room 354 Lab Phone: 541-346-5192. ... The primary techniques we use are mutagenesis, x-ray crystallography and spectroscopy, but ... Since 1996 we have worked with Green Fluorescent Protein, which spontaneously rearranges itself to ... Crystal structures were determined of related fluorescent proteins from corals that fluoresce yellow, orange...
12 September
The Haidekker Lab: Research Interests
A popular example is green fluorescent protein (GFP), which can be linked to gene expression and allows to study cell signaling and gene-related processes. ... Since fluorescence measurement is highly quantitative, fluorophores with sensitivity for environmental properties can act as high-speed, versatile, and accurate sensors. The UGA Biophotonics lab investigates novel applications of spectroscopic and laser-optical imaging techniques with a focus on fluorescent sensing.
10 April
Science Weekend Investigations and Field Trips
The fluorescent proteins (red, green or blue forms) produced by the bacteria will be collected and purified using chromatography and gel electrophoresis techniques. DNA and amino acid sequences published in the national database will be compared and analyzed. ... You will be learning and doing all of the steps from gene level to protein level. Approved students can sign up for the 1-unit BIOL 299 "Biological Sciences Independent Study" course, with Jo Wu's signature. Lab Techniques: lab safety, sterile techniques, Restriction enzyme digest, ligation, bacterial transformation, bacterial cultures, DNA...
11 December
A random approach to stabilize a
In this respect, the green fluorescent protein (GFP) fused to the intracellular C terminus of IMPs targets is a sensitive reporter that has enormously facilitated the precrystallization screening (Drew et al., 2006), Kawate and Gouaux, 2006), (Simon Newstead, 2007) and (Hammon et al., 2008). ... Most importantly, when GFP fluorescence is combined with size exclusion chromatography (fluorescence size exclusion chromatography, FSEC), it results in a powerful tool to characterize protein stability and quality under various conditions (e.g., homogeneity in different detergent solutions) using a very small amount of sample...
Predicting Cell Capture from Dilute
Mortality 20-28%. Rapid detection of bacteria. Technique. Features Live/Dead. Identification. ... Need to “see” cells for rapidly screening different filtration and sensing systems: led to use of GFP bacteria red or green E. coli green L. monocytogenes. (Applegate and Sedlak, 2004). Expressing Fluorescent Proteins. Fortineau et al. (2000) Res.
BIOPROBES 72
When a new user is trying to learn the specifics of the lab protocols in addition to the complexities of the equipment, frustration can set in. A microscope that is easy to use, while remaining flexible for a variety of techniques, is a welcome addition. ... The Click-iT Plus technology improves upon the first-generation Click-iT TUNEL Assays by the use of the picolyl azide combined with a copper protectant to reduce the exposure of fluorescent proteins, such as Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP), to the damaging effects of free copper.
Project: Protein Localization in
• Biological Techniques: – DNA extraction & purification – Transfection of DNA into HEK293T (human embryonic kidney). & NIH3T3 (mouse embryonic fibroblast) cells – Cell staining & fluorescence microscopy to visualize protein. location with GFP. Diseases associated with Protein Mislocalization. ... – Putting that DNA in mammalian cells (transfection) – Determining where our proteins are within the. cells (cell staining & fluorescent microscopy). Our Project Plan.
Ravi Palanivelu lab: May2006_Image of the month
To determine which cells within the female gametophyte expressed MYB98, they generated and analyzed transgenic A. thaliana plants containing ProMYB98:green fluorescent protein (GFP) constructs. Ovules from these plants were subjected to confocal microscopy analysis. This analysis showed that MYB98 is expressed only in the synergid cells until it is fertilized.
19 December
Biotechnology & Laboratory Science | Course Descriptions
Sterile laboratory technique, the preparation of different types of culture media, transformation, conjugation, and transduction of bacteria will be studied and practiced. Bacteria, bacteriophages, yeast, multi-cellular fungi, and nematodes will be cultured. Many molecular biotechnology techniques will be performed on microorganisms or using microorganism products. Restriction sites on plasmid DNA and Lambda virus DNA will be mapped. Bacteria will be transformed to make Green Fluorescent Protein (GFP); the Green Fluorescent Protein will be purified and analyzed...
1 October
Carl Zeiss Microscopy | Cyan and Green Fluorescent Proteins
Properties of GFP variants. High Performance Blue Fluorescent Proteins. Sirius (GFP Derivative). Emission Max = 424 nm Brightness = 3.6 Photostability ~ 75 Phenylalanine Chromophore. ... Emission Max = 454 nm Brightness = 44 Photostability ~ 60 LYG Chromophore. All BFPs can be imaged with DAPI Filter sets Omega QMax Blue Best for BFPs. Cyan and Green Fluorescent Proteins. mTurquoise & mCerulean3 (mCerulean Derivatives).
Purchase of a Fluorchem Imaging System
The desired Alpha Innotec system (Fluorchem HD2) allows chemiluminescent detection of Western blots, microarrays and macroarrays and fluorescent analysis of green fluorescent protein as well as detection of Coomassie and Ethidium bromide stained gels. ... Copper dependent enzyme abundance is measured at the DNA, RNA and protein levels to determine the cause of abnormal protein expression or activity. All of these techniques require functional imaging equipment. Perkins/Holy/Rose-Hellekant Laboratory.
Marine Biological Laboratory
Green fluorescent protein (GFP) purified by Osamu Shimomura from Aequorea. GFP glows bright green when hit with ultraviolet or blue light, a property that allows scientists to use it as a glowing tag inside cells. Photo credit, Tom Kleindinst. Osamu Shimomura holds a test tube containing green fluorescent protein (GFP) in a water solution. He also holds a lamp that shines ultraviolet light, which causes the GFP to fluoresce bright green.
4 October
Transfection efficiency monitoring: Green fluorescent protein...
Green fluorescent protein in CHO cells. Mock transfected cells GFP transfected cells. ... lab-on-a-chip technology can serve as a standardized approach to gel electrophoresis. ... Application: A comparative analysis of different techniques used for absolute protein.
Damer Lab Homepage - Lab Dicty Movies
Cynthia K. Damer's Research Laboratory. Department of Biology. Central Michigan University, Mount Pleasant, MI 48859. ... Wildtype cells incubated in water for 2 hours. DIC images were taken every 2 seconds QuickTime movie (30 sec.) A single cell expressing GFP-CpnA that has been starved for 5.5 hrs. GFP-CpnA translocates from the cytosol to membranes and back to the cytosol once.
9 December
Austin Community College, Biotechnology Department
Lab Competency Your competency in all the techniques in these lab exercises is the most important outcome of this class. Your ability to perform tasks successfully and use good lab technique will affect your grade. Your instructor will indicate on your graded report whether you have shown competency in these areas. ... 150. Lab unit 9: green fluorescent protein purification. (adapted from BioRad’s GFP Chromatography kit). OBJECTIVES Your performance will be satisfactory when you are able to.
University of Wollongong
Green fluorescent proteins (GFPs) and GFP-like fluorescent proteins have found extensive use in molecular and cellular biology [1–4]. Recently, photoswitchable fluorescent proteins have been reported [5–13] whose fluorescence properties can be altered upon illumination at specific wavelengths. ... This technique relies on the stochastic photoactivation and localization of single molecules (PALM, STORM, FPALM), in which a fluorescence image is constructed from the high-accuracy localization of individual fluorescent molecules that are switched on and off optically [16– 20].
Unveiling and blocking the interaction between tomato spotted
To achieve these goals, I expressed soluble and insoluble forms of GN fused to green fluorescent protein (GFP) transiently and transgenically and examined their cellular localization in planta. ... N aggregates (homopolymerization) are formed through homotypic interactions between N monomers as has been determined by yeast two-hybrid system, size-exclusion chromatography and fluorescence resonance energy transfer (FRET) techniques (Uhrig et al., 1999; Kainz et al., 2004; Snippe et al., 2005; Ribeiro et al., 2009a).
In this lab you will: Determine the yeast cell concentration in liquid culture using a hemocytometer and light microscope. Calculate the dilution factor necessary to dilute the culture to 50,000 cells/ml. Dispense and spread the cells on plates using proper sterile technique. ... The yeast you are using for these experiments contain a DNA repair protein, called Rad22, which is tagged with green fluorescent protein (gfp).
DOCX
Imaging | 1.1 Advantages of fluorescence detection
Green fluorescent protein and its variants Green fluorescent protein (GFP) is widely used as a reporter molecule for the study of protein localization, protein binding events, and gene expression (12). ... 1. Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Second Edition, Plenum Publishing, New York (1999). 2. Haugland, R. P., Introduction to Fluorescence Techniques, in Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc., Eugene, OR, pp. 1–4 (1996).
Application of GFP for bioprocess monitoring and control
Dr. Rao's lab: Another lab exploiting GFP's quantitative applications, with expertise on fluorescence technology and biosenor. Website under construction. ... Albano CR, Randers-Eichhorn L, Bentley WE, and Rao G 1998. Green fluorescent protein as a real time quantitative reporter of heterologous protein production.
18 June
Reports
Dirui Li and Ming Wang National Animal Protozoa Laboratory, Key Laboratory of Zoonosis of the Ministry of Agriculture, College of Veterinary Medicine, China Agriculture University, Beijing, China. ... These vectors involve fusing a gene of interest with the GFP reporter gene. However, this approach may disrupt the topology and function of the target protein or inhibit GFP fluorescence.
SDSU College of Sciences
The page you were looking for does not exist, please make a selection from the menu on...
Links to the page contain: Directed Evolution Of Green Fluorescent Protein By A New …...
15 December
Basement Membrane Components in C. elegans
Elliott Hagedorn & David Sherwood Sherwood's Lab, Duke University. Abstract. Green fluorescent protein (GFP) and other genetically encoded fluorescent proteins provide a means to study gene expression pattern and protein localization in living tissues. Recently discovered GFP-like fluorophores and engineered variants have further expanded the fluorescent protein toolkit for in vivo imaging. Here we describe a technique using transgenic C. elegans that contain laminin or type IV collagen fused to the green-to-red photoconvertible fluorophore Dendra2, to track the fate of the basement m...
Bacterial transformation
Note that the plasmid contains the gene of interest, green fluorescence protein (GFP), with a T7 promoter sequence in front of the gene, in addition to the origin of replication (ori) and the ampicillin resistance gene (?-lactamase or bla). ... However, the bacteria that have taken up the plasmid (transformed) and express the ampicillin resistance gene product from the plasmid will be able to grow on media containing ampicillin while all the other bacteria will die; a powerful technique called selection.
Lillemeier Lab - Salk Institute for Biological Studies
The lab uses cutting edge super-resolution and dynamic fluorescence microscopy techniques (e.g. PALM and FCCS) in combination with traditional biochemical and molecular biological approaches to study the molecular patterns that regulate and are required for T cell activation and function. ... T cell receptor (TCR) molecules, labeled with enhanced green fluorescent protein (eGFP), form microclusters at the entire the contact site between T cell and bilayer.
6 October
Using Encapsulated Fluorescent Bioprobes to Detect...
These genomic analyses of plants may one day provide a range of bio- and nanotechnologies for development to look for trinitrotoluene (TNT)-based materials using fluorescent or bright tags such as green fluorescent protein (GFP).2 These plant alterations will need to withstand the natural constraints of the environmental conditions, i.e., changes in soil pH. ... This minimal fluorescent yield can be reduced with further experimental technique that shuts down the fluorescence spectral signals; this is related to the bioprobe manufacturing process.
16 January
KEYWORDS: Green fluorescent protein, transformation...
transformation, and the convenience of using green fluorescent protein (GFP) as a molecular tag for studying cellular processes. The Bio-Rad kit comes with a recombinant plasmid called pGLO that replicates in E. coli and carries a gene (gfp) that encodes the GFP of the bioluminescent jellyfish Aequoria victoria (Shimomura, 1998). When GFP is illuminated with long wave ultraviolet (LW-UV) light it gives off a bright green fluorescence.
CMIC - Links | Fluorescence Microscopy
UCB, Electron microscopy lab - The EML is ... Protocols and Techniques [top] Laboratory Protocols ... Green Fluorescent Protein (GAP) - Wallace Marshall at Yale University has put together this extensive list of web-sites having to do with GFP applications and vectors.
9 February
Fluorescence Microscopy
Fluorescence microscopy in biomedical research is a light microscope technique designed to view fluorescence emission from a biological specimen. It has become an extremely useful tool to localize genes, messenger RNA and proteins within live and fixed cells and tissues and to visualize spatiotemporal variations in intracellular calcium as well as other ions and metabolites. ... Immunofluorescence microscopy and green fluorescent protein.
The University of New Mexico
GFP and related fluorescent proteins appear in over 50,000 publications and were the subject of the 2008 Nobel Prize in Chemistry. ... In another system we use two small pieces of GFP as tags and a third ‘detector’ fragment. Assembly can be programmed by attaching the small tags to interacting proteins, which drive the pieces together for detection by the third piece. We show how we and others use this suite of tools to solve problems in protein expression, monitor protein localization, and detect protein-protein interactions.
GFP: Lighting Up Life
GFP: Lighting Up Life. GFP and other fluorescent proteins have revolutionized biology because they allow scientists to look at the inner workings of living cells. ... Osamu Shimomura and Roger Y. Tsien for the discovery and development of the green fluorescent protein, GFP. GFP is widely used by biologists to visualize the development and function of cells. Mechanosensory Transduction and its Modification in C. elegans.
Domain by a green fluorescent protein
Thanks to Marianne McKee, Tiffany Jeanminette, Ryan Hay, Denny Dartez, Shannon O'Neal, and Bryan Perilloux for their valuable help in the lab. ... Chapter 8: ChBD-GFP Fusion Protein as a Chitin Specific Stain for Fluorescence Microscopy. ... 27 Table 3: Applications of Green Fluorescent Protein in Various Research Areas. ... "The application of fluorescent antibody technique to medical mycology: a review."
Sortwell Laboratory - Translational Science and Molecular...
A-H. Example of expression following unilateral (left side) intranigral injection of rAAV2/5 ?-syn mixed with green fluorescent protein (GFP) vector. ... His areas of technical expertise include molecular biology, immunohistochemistry, and behavioral evaluations. In the Sortwell Lab Chris focuses on the rodent deep brain stimulation project. In his spare time he likes to spend time with his kids, traveling, playing the ukulele, taking photographs and writing books.
9 February
Auxin Transport During Gravitropism and Phototropism using...
The images in this project make use of a reporter gene known as GFP, which stands for green fluorescent protein, a naturally occurring protein found originally in the jellyfish, Aequoria victoria. The cloning of the gene encoding this fluorescent protein made it possible to label virtually any gene product in vivo, without the addition of toxic dyes, opening up a whole world of possible experiments in cell and molecular biology. ... Images from the George W. Burns Scanning Transmission Electron Microscopy Lab.
14 December
The Nobel Prize in Chemistry | From Gene to Protein
The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2008 jointly to Martin Chalfie, Osamu Shimomura and Roger Y. Tsien, "for the discovery and development of the green fluorescent protein, gfp". Lessons from the jellyfish's green light Off the west coast of North America, floats the jellyfish Aequorea victoria. In its light-emitting organs resides the green fluorescent protein, GFP, which glows intensely under ultraviolet light.
Fluorescence microscopy evidence for quasi-permanent...
By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. ... This has been most convincingly demonstrated for the Type I AFP from winter flounder, for which both the binding plane and the direction of binding have been determined using a technique called ice etching (31). The binding plane defines a hexagonal bipyramidal crystal with a predicted c/a axial ratio of 3.3 to 1, a result that is consistently obtained for this AFP (32).
Sabanci University Research Database
Our screen consisted of ankyrin repeat plant proteins expressed in mammalian tissue culture cells along with an NF-kB reporter vector that expressed Green Fluorescent Protein (GFP) upon NF-kB activation. ... To perform our screen, we generated an NF-kB sensitive GFP reporter plasmid by standard molecular biology cloning techniques and we constructed a library of mammalian expression vectors that contained 90 A. thaliana cDNAs by Cre-mediated recombination between plasmids.
4 December
Articles | Figure 2 | Correlative fluorescence and electron
Morphology could be improved using traditional electron microscopy techniques, but fluorescent proteins are quenched by the acidic, dehydrated ... 14. Tsien, R. The green fluorescent protein. ... single concave microscope slides (SailingBoat Lab Co.), where.
Gene tag construction (gtc) module
Week 11: (GTC LAB 9) Protein induction and analysis 1) Induce production of tagged fusion protein with cadmium. ... Students will gain: • practice setting up reactions based on previous DNA quantification • practice applying E. coli transformation techniques. Time: Approximately 2 hours, including a one hour wait period. Materials: • pENTR/D-TOPO vector (Invitrogen) and cloning reagents (salt solution, water) • TOP10 chemically competent E. coli cells (Invitrogen) • LB + 50 !g/mL kanamycin agar plates • sterile glass beads for plating • used bead collection container • 0.5 mL microcentrifuge tubes (one per...
Study of cellular responses and protein interactions
2. Analyse cellular responses and interactions of A? using molecular and cell biology techniques 3. Develop in vivo assays to screen compounds that might competitively inhibit A? binding. The identification of cellular pathways affected by A? and the development of screening. ... Since this study utilises A? fused to GFP (a green fluorescent protein), protein expression was indicated by the fluorescence of recombinant cells. Previous studies in our lab have indicated maximal proportion of fluorescent cells and optimal protein expression at the log phase of cell growth.
A Synthetic Biological Engineering Approach to
Lastly, I would like to thank my fellow students who kept me company during all of the many hours studying and in the lab. ... 25. vii. 3. translocation of green fluorescent protein using a ... Synthetic biological engineering techniques and standard biological parts, called BioBricks, were used to carry out this study. ... GFP is a naturally-occurring fluorescent protein originally discovered in Aequorea victoria [46].
To further explore this role, our lab has turned to tools that can definitively elucidate hnRNP K’s translocation in-and-out of the nucleus, as well as directly quantitate its degradation rate, in vivo. An appropriate tool for such experiments is the monomeric Eos fluorescent protein (mEosFP), which can be stably and irreversibly photo-converted. This fluorescent protein naturally emits green light (~516nm) and upon photo-conversion with UV (~405nm), it stably emits red light (~581nm). Using traditional cloning techniques, we are fusing mEosFP (Xenopus codon specific)...
DOCX
Tissue Culture and Staining
permanent laboratory markers (any color(s), but must be water and alcohol resistant), lab coat. (optional). Course Description: The goal of this course is to give the student a solid foundation in the. theory and techniques of cell culture, preparing them for job placement in either academic or. ... DNA into cells. 3. Isolate cells from tissues to start primary cultures and propagate those cultures. 4. Freeze cells to store them indefinitely for later use. 5. Analyze chromosomes using In Situ Hybridization. 6. Detect and localize proteins in cells using immunostaining techniques.
Sindbis in Aedine Mosquitoes and Cell Lines
An example of a double promoter tag was done with SVHR, a heat resistant variant of AR339, with a green fluorescent protein (GPF) downstream of E1 from the Brown lab at North Carolina State University (Wang, 2008). ... In general the infection pattern follows established literature for wild-type SINV. The establishment of a titration protocol with fluorescent-tagged virus improves upon the standard plaque assay for a quicker and more accurate titration without moving to more complicated genomic techniques.
Rapid Spine Delivery and | GFP
GluR1 to 4 (GluRA to D) (7). A substantial proportion of endogenous AMPA receptors in hippocampal neurons have the GluR1 sub-unit (8). We constructed a recombinant GluR1 tagged with green fluorescent protein (GFP) at the putative extracellular NH2-ter-minus (GluR1-GFP; Fig. 1A) (9). This pro-tein was expressed in human embryonic kid-ney (HEK) 293 cells; extracts showed a sin-gle band by protein immunoblotting of the expected molecular mass (Fig.
Split-Superpositive Green Fluorescent Protein Reassembly...
The fusion of molecular biology techniques to control or alter protein sequence, and ... the last few decades is Green Fluorescent Protein (GFP), and is an excellent example of how protein ... Researchers in the Liu lab developed a positively supercharged variant of GFP...
Fluorescent-Antibody Techniques and ELISA
Fluorescent-antibody techniques use antibodies labeled with fluorescent dyes. ... Fluorescence is much easier and more sensitive for lab work but not practical for use outside of a well equipped lab. Western Blotting (Immunoblotting). Much like Southern blotting but uses enzyme linked antibodies to detect specific proteins rather than labeled probes to detect specific nucleic acid sequences.
23 March
DNA Science and Biotechnology | Brown University
Students will learn about DNA, RNA, and proteins and apply this knowledge to learning techniques used in the field of Molecular Biology. Students will manipulate DNA and express the Green Fluorescent Protein gene (normally found in the Pacific jellyfish) in bacteria and extract and observe both DNA and proteins using PCR (polymerase chain reaction) and electrophoresis. ... Environmental Leadership Labs.
14 November
Search Results
Green fluorescent protein (GFP). ... Green fluorescent protein (GFP). DSHB-GFP-8H11.
12 November
The Images from Science 2 Exhibition
Green Fluorescent Protein (GFP) has become an important part of biological investigations that requires non-invasive labeling of cells, especially in vivo. Derived from the jellyfish Aequorea victoria, GFP can be integrated directly into the DNA of a cell where it will naturally emit green fluorescence when excited with the proper blue wavelength. The fluorescence that is produced can then be used as a marker to follow a variety of biological events, from aging and cancer development to protein synthesis and xenotransplantation.
26 January
Absorption spectroscopy of the green fluorescent protein...
It is demonstrated that the storage ring technique with long storage times and suitable particle detection is ideal for studies of the photo-physics of chromophores. Here we discuss the absorption bands of the anion and cation chromophores of the Green Fluorescent Protein (GFP). We compare our gas-phase absorption measurements with absorption proles of the protein and of chromophores in liquids.
ISTANBUL
FM : Fluorescence miroscopy. GFP. : Green fluorescent protein. ... 1.7.4 Fluorescent tagging for labeling Immunolabeling and genetic tagging are general techniques for tagging of proteins. Using labelling techniques researchers can combine fluorescent techniques and biomolecules. For the detection of endogenous proteins the sample is labeled with a primary antibody and then amplified with a secondary antibody conjugated to a small organic dye or a QD.
SLU Physiology lab - sensory Demos - Stark
The 2004 physiology lab class collected pilot data suggesting that they do not. Then, an undergraduate student, Michael Haskins, working in my lab, followed by obtaining the data to prove this finding. His work earned him second place in biology's 2006 undergraduate research award, a monetary award, and recognition in the precommencement program and the publication of a research note (Drosophila Information Service volume 89 pp 17-19, 2006, on line). ... Demonstration of rhodopsin - metarhodopsin conversions and green fluorescent protein (GFP).
8 December
NIST
All Associates will receive a stipend increase after one year of tenure. Additional Information About National Institute of Standards and Technology (NIST). Prior affiliation includes direct full-time employment relationships with the laboratory. Research contracts with universities that provide support for graduate students or faculty who perform research on campus are not ordinarily considered to be disqualifying.
28 January
BMIL Data Sheet | Department of Biomedical Engineering
GFP. Green fluorescent proteins are being used for more and more applications in molecular and cellular biology. As a result of the variety of applications several variants form the original wild type green fluorescent protein (wtGFP) have been developed. ... Absorption and fluorescence emission spectra of fluorescein goat anti–mouse IgG antibody in pH 8.0 buffer. Green microspheres [FluoSpheres® polystyrene microspheres, 15 µm, green fluorescent (450/480)].
29 August
High Efficiency Transformation of Loblolly
An engineered green fluorescent protein (m-gfp5-ER) gene under the control of the 35S Cauliflower Mosaic Virus promoter was used to develop a facile and rapid loblolly pine (Pinus taeda L.) transformation system via Agrobacterium tumefaciens-mediated transformation of mature zygotic embryos. ... The m-gfp5-ER gene produced bright-green fluorescence easily detectable and screenable in loblolly pine tissue 3-30 days after explants were co-cultivated with Agrobacterium.
Reed Bio 342 | Then Why Do Some Jellies Appear Green?
This complex is often referred to as the Blue Fluorescent Protein, BFP (Kendall and Badminton). ... Oxidation of colenterazine to colenteramide is triggered with Ca2+ binds to the protein, and light is produced. (b) Crystal structure of a GFP mutant (Kendall and Badminton). Then Why Do Some Jellies Appear Green? The second step in some cnidarian luminescence is the transfer of energy to the green fluorescent protein, GFP, which emits green light through the process of fluorescence.
30 November
Practical 3. Analyzing DNA fragments
(A) Digestion of your plasmid DNA pCMV-GFP: The plasmid pCMV-GFP consists of the cDNA encoding the green fluorescence protein (GFP) cloned into the ... Collect the tubes with the solutions and enzymes from the bench at the front of the lab and place ... Ethidium bromide is a fluorescent dye that intercalates between the stacked bases of DNA. ... 1. List three techniques you have learnt from this practical. 2. Paste your gel photo on the left...
Coral Fluorescent Proteins as Antioxidants
10], for which fluorescent proteins (FPs) are largely responsible [8]. FPs are abundant and diverse within anthozoans, ranging across four basic color types; cyan (CFP), green (GFP), red (RFP) and a blue/purple non-fluorescent chromoprotein [11,12,13], however their function within the holobiont remains undetermined and controversial [14,15]. ... 15. Cox G, Matz MV, Salih A (2007) Fluorescence Lifetime Imaging of Coral Fluorescent. Proteins. Microscopy research and technique 70: 243–251.
Green fluorescence was observed on the cell surface
(1. College of B iological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, China; 2. Department of Environmental and Plant B iology, Ohio University, A thens, OH 45701, USA ). Abstract: In order to determ ine the subcellular localization of a classical arabinogalactan - p rotein (AGP) from A rabidopsis, the AGP coding region was cloned and fused to a green fluorescence p ro tein ( GFP).
Characterisation of mutants
Some of them directly involved with me in my research and others standing on the sidelines and providing vital support. Firstly, I have to thank my supervisor Emma Whitelaw. After completing my Honours year in Emma’s lab, I was thoroughly excited at being able to undertake my PhD in her lab as well. ... My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes.
What Drives the Redox Properties of Model Green...
he unique properties of GFP exploited in novel bioimaging techniques have revolutionized many areas in the life sciences1,2 and have motivated numerous experimental and theoretical studies.3A7 To better understand the rich and com-plex photophysics of GFP, one can analyze its properties from bottom up, that is, from understanding the intrinsic properties of isolated FP chromophores to their. ... (3) Zimmer, M. Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior.
Green period
Green period. v Fluorescent labels: varied, sensitive. v Multiple labeling. v Confocal microscopy, 2-photon excitation microscopy. v Genetic engineering. Green period. v Immunouorescence. v Intracellular injection of uorescent. dyes. v Transgenic expression of GFP linked to specic proteins. v Transplant of green stem cells. Immunouorescence: Calbindin + Kv3.4. Intracellular injection of a uorescent dye. Sekirnjak et al (2003).
Detecting g-protein coupled receptor interactions using
In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. ... Dr. Jeffrey M. Becker, as he gave me the opportunity to carry out my study at University of Tennessee, Knoxville. And also the members of Becker?s lab for sharing their great knowledge and experience with me without any hesitation. I owe sincere thanks for their hospitality and making me feel like a member of their lab.
Tulane University - School of Medicine - Lab Services...
Fluorescent Scopes. ... Location: JBJ Room 334 Price: $25/hour for unassisted use and $50/hour for assisted/training use Capabilities: • Imagine green, blue, red, and far-red fluorophores (GFP, Alexa 488/568/647).
14 August
Molecular Biosciences | Lab Address
Website URL. Lamppa Lab. Education: Reed College BA Biology, 1973 University of Washington Ph.D. Plant Biology, 1980. Research Summary: Organelle biogenesis in eucaryotes depends on the import of nuclear-encoded proteins from the cytosol. ... Low levels of SPP severely affect other steps in the import pathway, as demonstrated by following the fate of green fluorescent protein (GFP) fused to a transit peptide in the same transgenic plants.
23 January
Huntington Public Schools, NY
This lab technique is the basis for DNA fingerprinting which is used for a variety of applications including forensic analysis." The second lab focused on bacterial transformation, which in layman's terms is a type of genetic engineering. "In this lab, a plasmid vector was used to introduce a new gene into bacteria," Mrs. Beck said. The gene introduced into the bacteria was the green fluorescent protein gene that originates from a Pacific jellyfish.
Student Success - Ian Woods - Ithaca College
Specifically, neuronal outgrowth and branching morphology are quantified in embryos in which each candidate gene is overexpressed simultaneously with a fluorescent marker, driven by ... We have optimized the in situ hybridization techniques and begun colocalization ... For five of the cart genes, our lab has generated stable transgenic fish that allow ... Neuropeptides, small proteins that help neurons communicate, are implicated in...
18 July
Olson Laboratory | GFP GFP expression in a mouse embryo.
GFP Muscle-specific GFP expression in Drosophila melanogaster. Saffranin O Growth plate stained with Saffranin O. Prx_Cre Expression of Prx-Cre in limb buds. ... LacZ Expression of lacZ in developing skeletal muscle. Hearts Normal and hypertrophic hearts. GFP GFP expression in a mouse embryo.
16 December
Use of Fusarium oxysporum Schl. cucumerinum Transformed...
In this study, we constructed green fluorescent protein (GFP)-labeled Fusarium oxysporum strain FSY0957, which was isolated from cucumber wilt. ... The results showed that the colonization ability of GFP-labeled strain to the cucumber plant was higher than that to muskmelon and watermelon. We established a genetic transformation system to investigate the infection mechanism of wither pathogens and colonization of plant rhizosphere.
15 February
Current Research - Portera-Cailliau Laboratory
Inside the Portera-Cailliau Lab in early 2015. ... Our research employs a variety of techniques ranging from molecular biology to calcium imaging. ... We image neurons that express fluorescent proteins like GFP in transgenic mice or in electroporated mice.
7 September
The Role of Actin Binding Proteins in Cell Motility
their corresponding drug resistance are summarized in Table 1. Constructs of proteins fused to green fluorescent protein and red fluorescent protein are denoted by GFP and RFP respectively. ... His kindness and mentorship have proven invaluable, as he guided me through new scientific concepts and scientific techniques. I would also like to extend thanks to current and past Knecht Lab members, Evan Byron, Allie Goetjen, Nicholas Minutolo, Alex Marrotte, Gaurav Joshi, and Dr. Renee Gilberti for their friendship and assistance within the laboratory.
Constructing Copine A Mutants in... - Vassar College
This organism is unicellular and eukaryotic, and shares many of the same membrane trafficking pathways with mammalian cells. To determine the location of Copine A in Dictyostelium, we expressed a green fluorescent protein (GFP) tagged version of the Copine A protein (GFP-CpnA) in wildtype cells. Examining these cells under fluorescence microscopy we were then able to detect the location of the protein in the cell.
15 December
Introduction: The green fluoresent protein( GFP ) was made...
GFP is unique among light-emitting proteins in that it dose not require the presence of any cofactors or substrates for the generation of its green light. ... Importantly, detection does not require fixation or permeabilization of cells; therefore, compareed with immnuocytochemistry techniques using fixed cells, the likelihood of artifacts is reduced. Its tight, barrel-like structure protects the overall conformation of GFP protein and ensures that attachment of even large protein moieties does not affect its fluorescence capacity.
19 December
Understanding SNARE protein mediation of neuromodulator...
The Lochner lab employs molecular cloning techniques, disruption of gene expression, and super-resolution fluorescence microscopy to identify key proteins involved in the secretion of neuromodulators from DCVs. ... To better understand the molecular determinants of synaptic plasticity, my laboratory uses fluorescence microscopy and green fluorescent protein (GFP) technology to study the synaptic localization, distribution and secretion of tPA and other key neuromodulatory proteins at synaptic sites.
10 September
Vogel, Bruce | University of Maryland School of Medicine
Where is hemicentin expressed? We have constructed a functional fusion between hemicentin and green fluorescent protein (GFP), enabling us to monitor hemicentin-GFP localization in live animals during development. ... Lab Techniques and Equipment.
9 November
Doctor Anya Salih
Salih, A. (2012), 'Fluorescence control in natural green fluorescent protein (GFP)-based photonic structures of reef corals', Optical Biomimetics: Materials and Applications, Woodhead 9781845698027. Salih, A., Wormell, P., Garbutcheon-Singh, K., Harper, B., Myers, S., Geny, D., Hammang, C. and Aldrich-Wright, J. (2011), 'Applications of Fluorescence Spectroscopy and Confocal Microscopy.', Metallointercalators: Synthesis and Techniques to Probe Their Interactions with Biomolecules, Springer 9783211094273.
5 November
Calcineurin-NFAT Signaling Pathway Mediates NHE1 Induced
Osteopontin is a secreted protein involved in many physiological pathways including bone formation, immune response, vascularization and cardiac hypertrophy. The physiological pathway in which active NHE1 induces OPN expression in the heart remains unknown. Recently, NHE1 was described as a key regulator of the calcineurin-NFAT pathway. ... H9c2 cells were infected with active NHE1 or an adenovirus containing GFP only, as a control, using a multiplicity of infection 50 MOI. • Cell Area: Cells were visualized using inverted fluorescent microscope .
Computational | Average GFP after 300 20,000s = 488
• Due to time and expense of in vivo experiments, computational modeling a valuable tool. GFP Expression - no induction - averaged over 100 trials 100. 90 Average GFP from 0 to 20,000s = 54 Average GFP after 20,000s = 42. 80 Average over total time = 45. ... Fluorescence intensity (a.u.) IPTG aTc.
Confocal Laser Scanning Microscopy
When donor and acceptor dyes are close (2-6 nm), as when the intron is folded, energy transfer from donor to acceptor results in only the acceptor dye emitting light (red). From Zhuang Lab, Harvard University. COS-7 cells expressing a vesicular stomatitis virus G protein-green fluorescent protein (VSVG-GFP) chimaera. The VSVG-GFP is retained in endoplasmic reticulum (ER) membranes.
Electronic Excitations of the Green Fluorescent Protein...
Abstract Green fluorescent protein (GFP) is of interest due to its wide range of applications in molecular genetics, biochemistry and cell biology. GFP is produced by the jellyfish Aequorea Victoria and fluoresces in the green portion of the visible spectrum. The gene for GFP has been isolated and can be used to make an expressed protein fluorescent by inserting it into a genetic code, thereby giving a method of tracking it in living animals.
Mitochondrial Dynamics and Apoptosis during Mouse
...techniques, and we are analyzing population characteristics by Fluorescence Activated Cell ... A mitochondrial-GFP ES cell line has been developed in our lab as an imaging tool. ... behavior in transgenic mice with introduced green fluorescent protein (GFP).
Murphy Lab | Data
Murphy Lab - Data - UCSF yeast GFP image collection. ... W.-K. Huh, J.V. Falvo, L.C. Gerke, A.S. Carroll, R.W. Howson, J.S. Weissman & E.K. O'Shea, Global Analysis of Protein Localization in Budding Yeast, Nature, 425, 686-691 (2003).
13 December
Fluorescence Workshop | Absorption Properties of Proteins
Absorption. Emission. Intrinsic Protein Fluorescence. Fluorescent enzyme cofactors that bind to proteins: Examples: NADH. Riboflavins. ... The past decade has witnessed an explosion in the use of the family of naturally fluorescent proteins known as Green Fluorescent Proteins or GFPs. GFP, a protein containing 128 amino acid residues, was originally isolated from the pacific northwest jellyfish Aequorea Victoria.
Department of Biology : University of Vermont
To show that GFP is expressed by the bacteria using a Western blot. Module 3 Screening a Plasmid Library Through Protein Expression. This module lets you search for a gene of interest and demonstrate to us that you retrieved the right gene ?not unlike the process that you would go through in the lab. ... Analysis of PCR products by agarose gel electrophoresis for the Alu insertions in chromosome 8 and a polymorphism called a variable number tandem repeats (VNTR) D1S80 from the noncoding region of chromosome 1. Use the techniques you learned to help solve a ?crime?.
25 June
Two-photon directed evolution of green fluorescent proteins
Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies.
12 December
Biol 230 lab (cumulative review)
UV light (or Black light = long wavelength violet) was used to visualize colonies expressing Green Fluorescent Protein (produced when PBAD is activated by the proper Arabinose enantiomer.). 9. Ex. ... o Extracting macromolecules – DNA, Protein · Comparing Chromatography/separation techniques: paper, column (Gel Filtration; affinity/exchange/HIC; fluid phase, solid phase), electrophoresis. · Know your D1S80 Genotype!!!! Do the calculations, if you have not already!
Identification and characterization of IBR5
ACKNOWLEDGMENTS I would like to acknowledge the members of the Dharmasiri Lab, especially ... GFP - Green Fluorescent Protein GST - glutathione S-transferase; a fusion tag for ... Pull down assay - A technique to isolate and identify interacting proteins with a...
Evolutionary classification of protein domain
Lab Homepage. ... Gfp. Green fluorescent protein. Tree view.
27 January
What to do with a fluorophore | Green Fluorescent Protein
Green Fluorescent Protein imidazolidinone ring. GFP transcriptional reporter. ... The fluorescence emission spectrum (green) has a peak at 509 nm and a shoulder at 540 nm. GFP transcriptional reporter. 10. Green Fluorescent Protein. No Caption Found. J. Lippincott-Schwartz et al., Science 300, 87 -91 (2003) Published by AAAS.
Red Fluorescent Protein Protein
These proteins (mainly Green Fluorescent Protein) have been used extensively for labeling other proteins. While GFP has been extremely useful for labeling, the search for a protein that emits light at a longer wavelength has led to the discovery of DsRed. ... This leads to the formation of a GFP fluorophore, and green light is emitted.(13) The last stage of maturation occurs when the alpha carbon (Gln66) undergoes cis isomerzation and the amid nitrogen(Gln66) undergoes oxidation. The now mature fluorophore fluoresces red.
20 April
Mosaic Analysis with Double Markers (MADM)
The MADM target vector possesses transgenes targeted to the ROSA26 locus on copies of chromosome 6. The RG strain contains the sequence coding for the amino (N) terminus for green fluorescent protein (G... ... As referred to earlier, MADM recombination results in the construction of two full proteins, GFP and RFP. As the technique currently stands, the native GFP can easily be seen under fluorescence microscopy. However, RFP is too dim to be visualized without enhancement.
Fluorescent protein, green fluorescent protein, CFP, YFP...
Fluorescence resonance energy transfer (FRET) is a technique to resolve the proximity of proteins beyond the optical limit of light microscopes (in the range of 1–10 nm). It can be used to reveal inter-actions between proteins or structural changes within a molecule. ... rophores suitable for FRET experiments since besides other prerequisites the donor emission spectrum has to overlap the excitation spectrum of the acceptor. Fluorescent proteins (FPs) like the green-fluorescent-protein (GFP) are very attractive for FRET experiments.
Characterization of a Transgenic Pig Model Carrying Green...
Unfortunately, little is known about the interactions of proteasomal subunits with other sperm proteins or structures. Through a joint effort between Dr. Prather, Dr. Wells, and Dr. Sutovsky’s labs, a novel transgenic pig model with Green Fluorescent Protein (GFP) fused to the 20S proteasomal core subunit ?-type 1 (PSMA1) was created, hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes.
Molecular Cloning | Fusion proteins
– One xample is the green fluorescent protein or GFP. ... • Fluorescent proteins. – Examples is the green fluoresecent protein or GFP. • Epitope tag. – Short peptide sequence which has an antibody that recognizes it specifically. Type of Tags. • Fluorescent proteins.
Exam Mol 3008 – May 2009
19. Green fluorescent protein (GFP) has become an important tool in life science. ... What is this technique based on? A The irreversible photo-bleaching of a fluorescent protein B The reversible photo-bleaching of a fluorescent protein C The intensity of green-fluorescent protein (GFP) D The mobility of a particular fusion protein E Rapid image acquisition of live cells.
Short Technical Reports
CEM-GFP cells, containing a reporter green fluorescent protein (GFP) under the control of HIV-1 long terminal repeat (LTR), were electroporated with pCMV-Tat encoding the viral transactivator protein. ... Essentially identical results were obtained with RNA extraction columns from a different vendor (Auprep; Life Technologies, Invitrogen; data not presented). RFU, relative fluorescence units; A, absorbance.
Fluorescence Resonance Energy Transfer Microscopy
In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behav-ior of expressed fusion proteins. The genetic engineering of jelly-fish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum.
Plant cell imaging
Welcome to the Ehrhardt laboratory at the Carnegie Institution for Science. We investigate plant cell development and morphogenesis, utilizing live cell imaging techniques to visualize the organization and dynamic behaviors of molecules and organelles. Problems of special interest include how cells generate asymmetries and specific shapes. Currently we are investigating how the cortical microtubule cytoskeleton acquires organization and how this organization in turn functions to guide patterns of cell growth and division.
10 July
Nucleic Acid Origami
The mathematical technique known as wavelet analysis is useful for everything from ... The Gene Expression Database, hosted by the Jackson Lab-oratory in Bar Harbor ... Green fluorescent protein (GFP) has allowed researchers to peer into the bustling world...
San Jose State University
The first aim of this study was to test whether DPE2 translocates to a microsomal compartment in response to cold by both a biochemical approach (western blot) and a cellular approach (green fluorescent protein tagging and confocal microscopy). ... I am indebted to Dr. Zhiping Deng from the Wang lab at Carnegie for guiding me with my experiments and training me in plant biology techniques. I also wish to thank all my friends at the Wang lab for their support and for providing a stimulating environment in which to learn.
College of Pharmacy - Pharmaceutical Sciences - Lentiviral...
pLenti CMV GFP Blast vector: Contains a cytomegalovirus (CMV) promoter driving expression of enhanced green fluorescent protein (EGFP), as well as a safety modified woodchuck hepatitis post-transcriptional regulatory element (WPRE,W). This vector also contains a Simian vacuolating virus 40 (SV40) early promoter and the prokaryotic EM7 (EM7) promoter driving expression of a blasticidin (Blast) resistance gene. Trobridge Lab - pRSC-SChW.
7 February
Microscope Imaging Station. Gallery.
Nematodes (Caenorhabditis elegans) that express the gene for green fluorescent protein (GFP). Three distinct expression patterns for GFP are visible in this population of worms. This movie plays in real time. Elapsed time 50 seconds. This movie was taken on an upright dissecting microscope with a fluorescent lightsource, 10x objective GFP filter set and digital video camera. Fluorescent light and background transmitted light were used simultaneously.
7 April
Biol 189T
Small molecules, Proteins. ... Lab note books due. Schedule is tentative, any changes will be announced in class. ... sterile techniques. 29.
25 February
Microbiology Lectures : MOLB 2210
Transformation allows bacteria to make a protein (or proteins) that give them ... In nature, the Regulatory C protein stops the production of the enzymes needed to digest the simple sugar ... by the gene encoding for the Green Fluorescent protein.
10 December
Subproject E4.01
FCS is a widely used technique to characterize the dynamics of fluorescent species ... We have also developed lab-on-the-chip devices that allow fast mixing and exchange of ... induced protein cleavage and green-to-red conversion of fluorescent protein EosFP...
Inside
This vector contains a stability region so that expression of its gfp is more stably maintained within bacterial cells in the absence of selective pressure [6]. The half-life of the GFP protein is approximately one day [32] and so green fluorescent cells are considered to be metabolically active in situ and can be observed for a long time due to the. ... Nature 417: 962-966. 12. Fahraeus, G. 1957. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J. Gen. Microbiol.
Fluorescent Proteins Expressed in Mouse Transgenic Lines
Fluorescent proteins of the green fluorescent protein (GFP) fam-ily ... Transgenic mice were generated using standard techniques previously described ... aperture (NA) 0.75] or 63? (NA 0.9) water immer-sion objectives, and a MicroMAX 5 MHz cooled CCD camera attached to a Macintosh computer running IP Lab software. ... Huang WY, Aramburu J, Douglas PS, Izumo S (2000) Transgenic expres-sion of green fluorescence protein can...
Original Article
Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343 bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter.
Choosing Filters
of the protein of interest. Complete separation of. and green fluorescent pro-. the fluorescent signals re-. tein (GFP) or its variants. quires careful selection of. The fusion construct can. ... CELL ANALYSIS. Figure 5. Emission fingerprinting reveals complete bleaching of yellow fluorescent protein and concomitant dequenching of cyan fluorescent protein, indicating fluorescence resonance energy transfer in the bleached areas (marked by dashes). the range of 1 to 10 nm).
Genome Editing
induced Pluripotent Stem Cell Green Fluorescent Protein CRISPR Associated protein 9 ... Many cell types are not able to be cultured in the lab and often times the cell lines that are ... Immunostaining: This technique utilizes fluorescent antibodies to label specific...
Green Fluorescent Protein | Center for Advanced Microscopy
Techniques. ... Green Fluorescent Protein (GFP) was originally isolated from the jellyfish Aequorea victoria and is used to fluorescently label cells and organisms, as well as specific proteins when incorporated into a chimeric “fluorescent fusion protein”. Subsequently, numerous variants of GFP with altered characteristics, such as shifted excitation and emission spectra, have been generated, and fluorescent proteins from a variety of other organisms have been identified.
1 September
DigitalCommons@IMSA - IMSAloquium Student Investigation...
Commercially available NMDAR subunit specific antibodies from Cell Signaling and Abcam were shown to not be very specific and can skew results. The purpose of this investigation was to create a green fluorescent protein (GFP) tagged NR2B expressing vector that could be used to more accurately measure NMDAR2B trafficking. Following the successful creation of the GFP tagged NMDAR2B vector, the transfection protocol for this specific vector was optimized.
31 May
Lab 2: Microscopy: brightfield, phase-contrast, fluorescent...
1/26: Lab 1: Introduction t o laboratory techniques: pipeting, calculations, introduction to laboratory eql-~ipmenat nd lab safety, sterile technique. 2/2: Lab 2: Microscopy: brightfield, phase-contrast, fluorescent, and electron. ... 3/23: Lab 6: Colutnn chromatography. 3/30: Lab 7: Protein electrophoresis: 1D polyacrylamide gel electrophoresis (PACE) and examples of 2D electrophoresis.
Transgenic plants and animals: Examples
Many stable transgenic zebrafish lines now express jellyfish green fluorescent protein (GFP) genes under tissue-specific promoters. Because GFP expression can be observed in live embryos and adult, these transgenic zebrafish provided a powerful tool in developmental analysis. work of Gong Zhiyuan Department of Biological Sciences, National University of Singapore.
17 September
First molecular identification of the transgene red fluorescent...
In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio), found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP) transgene was found to determine different gradients-of-bioluminescence (shades in color) in each GMO fish analyzed. ... Gong, Z.; Ju, B.; Wan, H. 2001. Green fluorescent protein (GFP) transgenic fish and their applications.
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