Navigation Links
Monitoring fluorescence resonance energy transfer (FRET) between GFP,,, fusions in lysates of the yeast Saccharomyces cerevisiae using the ,,, Varian,,, Cary Eclipse

Monitoring fluorescence resonance energy transfer (FRET) between GFP
fusions in lysates of the yeast Saccharomyces cerevisiae using the Varian
Cary Eclipse

Paul Gavin# and Mark Prescott#, Ph.D

Daren J. Fyfe, Ph.D*

# Department of Biochemistry and Molecular Biology, Monash University, Clayton campus Victoria 3800, Australia

* Technical assistance: Varian Australia Pty Ltd, Mulgrave, Victoria 3170, Australia E-mail: fluorescence@varianinc.com

Introduction

Fluorescence resonance energy transfer (FRET) is a non-destructive, spectroscopic approach that can be used to monitor the proximity and angular orientation of donor and acceptor fluorophores in living cells1. The resonant energy of an excited donor fluorophore (in this example blue fluorescent proteinBFP) is absorbed by an acceptor fluorophore (green fluorescent proteinGFP) providing that donor and acceptor are in close proximity (between 1080 ngstroms2) (Figure 1). Emission spectra of donor fluorophores must significantly overlap the absorption spectra of the acceptor, while overlap between respective absorption and emission spectra of donor and acceptor should be minimized 3 .

Due to the attractive attributes of GFP (previously described in fluorescence application note No. 5)4, the application of FRET to GFP and GFP variants has become a powerful tool to monitor interactions at the protein level, within an intact cell or organism. It is possible to use FRET to measure conformational changes in a molecule tagged with two GFP variants in response to the binding of ligands such as calcium1, or the interaction between separate proteins, each tagged with a specific GFP5. Studies such as these present the unique opportunity to study subtle relationships and dynamic interactions between proteinsin living cells.

The present study aimed to detect and monitor changes in FRET between BFP and GFP in cytosolic lysates of yeast cells using the Varian Cary Eclipse.

Materials and Methods

Equipment

(For part numbers see reference6)

Varian Cary Eclipse fluorescence spectrophotometer

Peltier-thermostatted multicell holder (with electromagnetic stirring)

Temperature controller

Temperature probes

Magnetic stirrer bars

Yeast strains

YRD15 (MATa, his3, ura3, leu2, p+) of the yeast S. cerevisiae was the parental strain used in this study. A gene cassette was constructed encoding BFP and GFP linked by a 27 amino acid peptide linker that contains a recognition site for the protease trypsin. This cassette was cloned into the yeast expression plasmid pAS1N for cytosolic expression and transformed into the yeast strain YRD15 as previously described7. Transformants were plated out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids, 2% glucose, 1.5% agar) with growth supplements as required and grown at 28C for 35 days.

Protocol

Yeast cells were washed twice in 1ml MilliQ water before being lysed using Y-PER (Progen) as per the manufacturers instructions. Lysates were preferred over whole cells to allow protease digestion of the peptide linker. Y-PER lysates (10l) were diluted with 1.2ml Tris/HCl pH 8 and placed in disposable fluorescence cuvettes (Sarstedt) of the multicell holder positioned within the sample chamber of the Varian Cary Eclipse. The temperature within the cuvettes was set to 25C to promote cleavage of the peptide linker by trypsin (Figure 2). Using the Scan application, BFP was specifically excited using light of 360nm, and emission spectra for the fusion protein were recorded from the range 400550nm. Further emission scans were recorded over time after the addition of 0.25g trypsin.

Results

Emission spectra of the BFP-GFP fusion protein following 360nm excitation are shown in Figure 3. An initial spectrum was taken at time = 0 min, then trypsin was added and spectra were recorded at the times indicated. GFP emission (~510nm) is seen upon specific excitation of BFP alone (360nm), indicative of FRET. Spectral characteristics of FRET (indicated by the green peak at 510nm) progressively disappeared following the addition of trypsin, which cleaves the peptide linker that tethers the GFPs. A small increase is seen in BFP emission as FRET diminishes.

Discussion

An ideal strategy to monitor the interactions of proteins in living systems involves detection of FRET between BFP and GFP bound to target species of interest. For optimum selectivity and sensitivity of detection of fluorescence in applications such as this it is necessary to minimise (a) detection of cellular autofluorescence and (b) photobleaching. These issues are addressed by internal filters (on both excitation and emission monochromators) and the Cary Eclipse xenon flash lamp respectively. The issue of photobleaching is extremely important and is discussed in a separate application note.

The data indicate (Figure 3) that FRET could be accurately monitored in cytosolic lysates from yeast cells. Cleavage of the BFP-GFP fusion using trypsin (Peltier temperature controlled at 25C) demonstrated that GFP emission was due to FRET and not to direct stimulation of GFP by the excitation wavelength. This is demonstrated by the scans shown in Figure 3 that depict the green (FRET) peak becoming smaller (as BFP is cleaved from GFP) with successive scans over a period of 33 minutes.


Conclusion

Conclusion The Varian Cary Eclipse with Peltier temperature control and multicell accessories provide a simple and accurate assembly with which to monitor cell function at the protein level in cytosolic lysates from yeast cells. The opportunity now exists to use this model as a platform with which to investigate protein-protein interactions in response to external or internal stimuli in living cells.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Versatile Reporter Vectors for Monitoring Viral Transduction
2. Monitoring transfection efficiency by green fluorescent protein (GFP) detection with the Agilent 2100 bioanalyzer
3. Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System
4. Monitoring Bacterial Genetic Diversity in a Freshwater Lake Using TTGE and DNA Sequence Analysis
5. Direct Monitoring of Solid-Phase Peptide Synthesis by MALDI-TOF MS
6. Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation
7. New statistical algorithms for Monitoring Gene Expression on GeneChip® Probe Arrays
8. Immunoassay of Theophylline by fluorescence polarisation
9. Comparison of Performance between the AlphaQuest - HTS and the Fusion α Multilabel Reader for Detection of cAMP
10. Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae: Detection in vivo using the Varian Cary Eclipse
11. Saccharomyces cerevisiae
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/4/2016)... ... February 04, 2016 , ... ... Forensics Club, takes place February 5-6 at the University’s student center, Kehr ... activities such as workshops and competitions for ample networking, learning and collaborating ...
(Date:2/4/2016)... 4, 2016  CytoSorbents Corporation (NASDAQ: CTSO ... flagship CytoSorb® blood filter to treat deadly inflammation ... world, announced that CEO Dr. Phillip Chan ... Capital Group,s 2016 Disruptive Growth & Healthcare Conference, ... Conference Presentation Details: Where: Convene ...
(Date:2/4/2016)... --> --> ... pleased to provide the following update on recent corporate developments. ... last 3 months we have significantly increased our cash position ... As a result, we have positioned ourselves to execute on ... and expect that development to continue on schedule. ...
(Date:2/4/2016)... 2016  Spherix Incorporated (Nasdaq: SPEX ) -- an intellectual ... of intellectual property, today provided an update on the ... District of Texas and announcing ... Inter Partes Re-examination ("IPR") proceedings that VTech and ... was initiated on only certain claims of two of ...
Breaking Biology Technology:
(Date:1/18/2016)... 18, 2016  Extenua Inc., a pioneering developer ... use and access of ubiquitous on-premise and cloud ... American Cyber.  ... transformational C4ISR and Cyber initiatives in support of ... proven technology solutions," said Steve Visconti , ...
(Date:1/13/2016)... 13, 2016 --> ... new market report titled - Biometric Sensors Market - Global ... - 2023. According to the report, the global biometric sensors market was ... to reach US$1,625.8 mn by 2023, expanding at a ... of volume, the biometric sensors market is expected to ...
(Date:1/11/2016)... 2016 Synaptics Incorporated (NASDAQ: SYNA ), ... that its ClearPad ® TouchView ™ 4300 ... separate categories in the 8 th Annual Mobile ... Breakthrough. The Synaptics ® TDDI solution enables faster ... thinner devices, brighter displays and borderless designs. ...
Breaking Biology News(10 mins):