Fluorescence recovery after photobleaching studies of lipid rafts.
Fluorescence recovery after photobleaching (FRAP) is a microscopy-based technique that can be used to ask how lipid rafts impact protein and lipid diffusion in cells. This chapter, describes how to perform FRAP measurements of putative raft and nonraft proteins and lipids using a confocal microscope. Methods have been outlined for (1) transfecting cells with plasmids encoding for the expression of green fluorescent protein-tagged proteins, (2) labeling cells with fluorescent lipid analogs or with the lipid-binding toxin cholera toxin B-subunit, (3) depleting and loading cholesterol into cell membranes using methyl-beta-cyclodextrin, and (4) performing and analyzing confocal FRAP measurements.
Kenworthy
Anne K
AK
Dept. of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, Nashville, TN 37232, USA.
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000737:chemistry; D002784:Cholesterol / Q000172:deficiency; D003198:Computer Simulation; D004058:Diffusion; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D014162:Transfection
D008563:Membrane Lipids; D008565:Membrane Proteins; D002772:Cholera Toxin; D002784:Cholesterol