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Ma-PbFRET: Multiple Acceptors FRET Measurement Based on Partial Acceptor Photobleaching

Published online by Cambridge University Press:  25 January 2013

Huaina Yu ,
Jianwei Zhang ,
Huali Li  and
Tongsheng Chen
Huaina Yu
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Jianwei Zhang
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Huali Li
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Tongsheng Chen*
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
*
*Corresponding author. E-mail: [email protected], [email protected]
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Abstract

Fluorescence resonance energy transfer (FRET) measurement based on partial acceptor photobleaching (PbFRET) is easy to implement without external references. However, the current PbFRET methods are inapplicable to the construct with multiple acceptors, which largely increase the Förster distance. Here, we proposed a linear theory for the dependence of the acceptor photobleaching probability of construct with multiple acceptors on the photobleaching degree (x) and developed a multiple acceptors PbFRET method (Ma-PbFRET) to measure the FRET efficiency of construct with multiple acceptors (n) by measuring the fluorescence intensities of both donor and acceptor channels before and after acceptor photobleaching. The Ma-PbFRET method was validated by measuring the FRET efficiency of construct with two or three acceptors under different x in living cells. Our experimental results demonstrate that the Ma-PbFRET method is capable of exactly quantifying the FRET efficiency of construct with multiple acceptors, providing a simple and powerful tool to investigate the assembly/disassembly of biomolecular complexes with larger distance in living cells.

Keywords

FRETpartial acceptor photobleachingphotobleaching degreephotobleaching probabilityliving cellsdonoracceptor
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 171 - 179
Copyright
Copyright © Microscopy Society of America 2013

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