TY - JOUR
T1 - Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP)
AU - Day, Charles A.
AU - Kraft, Lewis J.
AU - Kang, Minchul
AU - Kenworthy, Anne K.
PY - 2012
Y1 - 2012
N2 - Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by stepby-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented.
AB - Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by stepby-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented.
KW - Confocal laser-scanning microscopes
KW - Diffusion
KW - FRAP
KW - Fluorescence microscopy
KW - GFP
KW - Protein trafficking
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U2 - 10.1002/0471142956.cy0219s62
DO - 10.1002/0471142956.cy0219s62
M3 - Article
C2 - 23042527
AN - SCOPUS:84874147884
SN - 1934-9297
SP - 2.19
JO - Current protocols in cytometry
JF - Current protocols in cytometry
IS - SUPPL.62
ER -