KEYWORDS: Fluorescence resonance energy transfer, Green fluorescent protein, Proteins, Image analysis, Control systems, Calibration, Switches, Spectroscopy, Signal detection, Molecular interactions
Fluorescence Resonance Energy Transfer (FRET) spectroscopy is an ideal tool to assess protein-protein interactions in cells. FRET occurs if suitable fluorophores are less than 10nm apart and thus may be used to probe molecular scale interactions. To obviate the need for fixation and staining, the proteins of interest can be linked to Green Fluorescent Protein (GFP) variants, which can then be expressed in intact living cells. However, FRET is inherently difficult to validate and interpret in such environments, making suitable positive controls vital. In this study cyan and yellow variants of GFP (CFP and YFP) were linked by amino acid chains of known lengths (6, 14, 16, 18, and 22 amino acids). Ratiometric FRET measurements were obtained via the acceptor photobleaching technique. The data was analyzed by new FRET analysis software developed by this group, which will be made available to the public. The evaluation method is novel in that it assesses an entire field of view for FRET efficiencies, thus making high throughput data analysis possible. A perfect candidate for FRET studies is the cyclin-CDK switch, responsible for the regulation of mitosis. Preliminary results with CyclinB1-CFP and cdk1-YFP are presented.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.