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

een 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 mult
'"/>

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:
(Date:7/10/2014)... --  Ceres, Inc . (Nasdaq: CERE ), ... results for the three months ended May 31, 2014 ... Ceres reported that the company and its customers have ... in Brazil, which concluded in June, despite dry and ... for part of the growing season. Yields of several ...
(Date:7/10/2014)... June 17, 2014 Using microscopic polymer light ... gases, researchers at MIT,s Quantum Photonics Laboratory have ... in the parts-per-billion range. Optical sensors are ideal ... high signal-to-noise ratio, compact, lightweight nature, and immunity ... sensors had been developed before, the MIT team ...
(Date:7/10/2014)... effect in bilayer graphene and shown that this exotic ... field. , The fractional quantum Hall effect, which ... exposed to large magnetic fields, is a striking example ... as a single system. However, while the basic theory ... this collective behavior remain not well understood, in part ...
(Date:7/10/2014)... the 1960s, theatergoers have shelled out for crude ... enhance their viewing experience. These basic devices, used ... three-dimensional reality, may soon be rendered obsolete with ... Tel Aviv University researchers. , TAU doctoral students ... developed highly efficient holography based on nanoantennas that ...
Breaking Biology Technology:Ceres Announces Fiscal Third Quarter 2014 Financial Results 2Ceres Announces Fiscal Third Quarter 2014 Financial Results 3Ceres Announces Fiscal Third Quarter 2014 Financial Results 4Ceres Announces Fiscal Third Quarter 2014 Financial Results 5Ceres Announces Fiscal Third Quarter 2014 Financial Results 6Ceres Announces Fiscal Third Quarter 2014 Financial Results 7Ceres Announces Fiscal Third Quarter 2014 Financial Results 8Ceres Announces Fiscal Third Quarter 2014 Financial Results 9Swell new sensors 2Columbia researchers observe tunable quantum behavior in bilayer graphene 2Columbia researchers observe tunable quantum behavior in bilayer graphene 3Projecting a 3-dimensional future 2Projecting a 3-dimensional future 3
... 26 ChromaDex Corporation (OTC Bulletin Board: CDXC), ... innovative ingredients to the dietary supplement, food, beverage and ... This exciting new product introduction is based on the ... rights of pterostilbene from the University of Mississippi ...
... static. In our brains, information processing circuitsneuronsevolve continuously ... team from Japan and Michigan Technological University has ... an organic molecular layer that can solve complex ... "evolutionary circuit" has been realized. This computer ...
... MT. LAUREL, N.J. , April 23 ... provider of technology-enabled clinical documentation services, today announces that ... of Spheris Inc.  Simultaneously, CBay Inc., MedQuist,s majority owner, ... a subsidiary of Spheris.  Together, the combined companies will ...
Cached Biology Technology:ChromaDex Launches pTeroPure Pterostilbene 2ChromaDex Launches pTeroPure Pterostilbene 3ChromaDex Launches pTeroPure Pterostilbene 4ChromaDex Launches pTeroPure Pterostilbene 5MedQuist Announces Completion of Spheris Transaction 2MedQuist Announces Completion of Spheris Transaction 3
(Date:7/11/2014)... at the National Institute of Standards and Technology ... work more effectively, they often prove that necessity ... the case recently for M. Lorna De Leoz ... growing specialization of glycomics. Glycomics is the study ... that are attached to proteins and lipids and ...
(Date:7/11/2014)... July 3, 2014, Shenzhen, China Researchers from Salk ... for the first time evaluated the safety and ... and successfully developed a new method, TALEN-HDAdV, which ... pluripotent stem cell (hiPSC). This study published online ... important theoretical foundation for stem cell-based gene therapy. ...
(Date:7/11/2014)... from the Max Planck Institute for Medical Research in ... understanding photosynthesis, the process by which the Earth first ... and which is therefore crucial for all higher forms ... the first direct visualization of a crucial event in ... specific protein complex, photosystem II, splits water into hydrogen ...
Breaking Biology News(10 mins):A new genome editing method brings the possibility of gene therapies closer to reality 2A first direct glimpse of photosynthesis in action 2
... Polar Regions and the deep sea is the focus ... involving over 200 international scientists. The CAREX (Coordination ... roadmap outlines priorities for future research into life in ... Life has evolved thousands of strategies to adapt and ...
... nearly 390,000 chemical compounds had led scientists to a ... the brain with effects promising for new drugs for ... which blocks formation of cholesterol in the brain, in ... G. Kazantsev and colleagues previously discovered that blocking cholesterol ...
... lifted off on board India,s Polar Satellite Launch Vehicle ... on 20 April 2011. The X-SAT, developed ... collaboration with DSO National Laboratories, was launched from the ... India. The wholly made-in-Singapore satellite was one ...
Cached Biology News:Life in extreme environments paves the way for international collaboration 2Singapore's first locally made satellite launched into space 2
50 assays. Provides an easy system for rapid separation of cytosol and particulate fractions that are compatiable with downstream studies....
Eliminate electrical interference from any analog signal, without filtering. Ideal for electrophysiology applications like EKG, EMG, and EEGs. Removes 50/60hz noise and harmonics caused by grounding ...
... SizeSep 400 Spun Columns, 10. *SizeSep ... cDNAs > 400 base pairs in length. ... Sepharose CL-4B and pre-equilibrated in distilled water containing ... smaller than ~ 400 base pairs. ...
... 25 purifications. Allows rapid purification ... removal of unincorporated CyDye label and ... yields of labeled cDNA probe. ... labeled by either direct incorporation or ...
Biology Products: