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Proteins within most macromolecular complexes or organelles continuously turn over. This turnover results from association and dissociation reactions that are mediated by each of the protein's functional domains. Thus, studying organelle or macromolecular formation from the bottom up using theoretical and computational modeling approaches will necessitate the determination of all of these reaction rates in vivo. Yet current methods for examining protein dynamics either necessitate highly specialized equipment or limit themselves to basic measurements. In this protocol, we describe a broadly applicable method based on fluorescence recovery after photobleaching (FRAP) for determining how many reaction processes participate in the turnover of any given protein of interest, for characterizing their apparent association and dissociation rates, and for determining their relative importance in the turnover of the overall protein population. Experiments were performed in melanoma M2 cells expressing mutant forms of ezrin that provide a link between the plasma membrane and the cortical actin cytoskeleton. We also describe a general strategy for the identification of the protein domains that mediate each of the identified turnover processes. Our protocol uses widely available laser-scanning confocal microscopes, open-source software, graphing software and common molecular biology techniques. The entire FRAP experiment preparation, data acquisition and analysis require 3-4 d.

Type

Journal article

Journal

Nature protocols

Publication Date

05/2015

Volume

10

Pages

660 - 680

Addresses

The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Keywords

Tumor Cells, Cultured, Cell Membrane, Animals, Mice, Melanoma, Proteins, Microscopy, Confocal, Calibration, Fluorescence Recovery After Photobleaching, Protein Structure, Tertiary, Image Processing, Computer-Assisted, Software