Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching-based fluorescence resonance energy transfer measurements.
نویسندگان
چکیده
Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels.
منابع مشابه
Analysis of FRET signals in the presence of free donors and acceptors.
A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unles...
متن کاملFixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.
Fluorescence resonance energy transfer (FRET) is a technique used for the study of functional interactions between molecules. The intimate vicinity between two fluorescent molecules (FRET-pair; donor and acceptor) allows for an energy transfer, which can be directly calculated as the so called FRET efficiency. This technique is used in fixed as well as living cells. Here we show first, measured...
متن کاملAPPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy
BACKGROUND Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Am...
متن کاملVisualization of the Activity of Rac1 Small GTPase in a Cell
Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-...
متن کاملSensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples
BACKGROUND Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS To allow side-by-side comparison of several fluoresc...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Journal of biomedical optics
دوره 17 1 شماره
صفحات -
تاریخ انتشار 2012