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Conditions for using FRAP as a quantitative technique—Influence of the bleaching protocol

  1. Dominika O. Trembecka,
  2. Mateusz Kuzak,
  3. Jurek W. Dobrucki*

Article first published online: 3 FEB 2010

DOI: 10.1002/cyto.a.20866

Issue

Cytometry Part A

Cytometry Part A

Volume 77A, Issue 4, pages 366–370, April 2010

Additional Information

How to Cite

Trembecka, D. O., Kuzak, M. and Dobrucki, J. W. (2010), Conditions for using FRAP as a quantitative technique—Influence of the bleaching protocol. Cytometry, 77A: 366–370. doi: 10.1002/cyto.a.20866

Author Information

  1. Faculty of Biochemistry, Biophysics and Biotechnology, Division of Cell Biophysics, Jagiellonian University, Kraków, Poland

*Faculty of Biochemistry, Biophysics and Biotechnology, Division of Cell Biophysics, Jagiellonian University, Kraków, Poland

Publication History

  1. Issue published online: 19 MAR 2010
  2. Article first published online: 3 FEB 2010
  3. Manuscript Accepted: 5 JAN 2010
  4. Manuscript Revised: 23 DEC 2009
  5. Manuscript Received: 17 JUL 2009

Funded by

  • Ministry of Science and Higher Education. Grant Number: 2067/P01/2007/32
  • Jagiellonian University

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Keywords:

  • FRAP;
  • fluorescence recovery after photobleaching;
  • nuclear proteins;
  • histone;
  • mobile fraction;
  • protein dynamics

Abstract

Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent way of data analysis. © 2010 International Society for Advancement of Cytometry

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