Fluorescence Recovery After Photobleaching
This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP). To quantify the dynamics of proteins within a subcellular compartment, we first outline the general aspects of FRAP experiments and then provide a detailed protocol of how to measure and analyse the most important parameters of FRAP experiments such as mobile fraction and half-time of recovery.
Key wordsFluorescence recovery after photobleaching Fluorescence microscopy Green fluorescent protein Half-time of recovery Mobile fraction Diffusion Binding reaction kinetics Focal adhesions Vinculin
We would like to thank Dr. Janet Askari for critical reading of the manuscript. CB acknowledges BBSRC (BB/G004552/1). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.
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