Fluorescence Recovery After Photobleaching Studies of Lipid Rafts
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Last Update: 2021-03-03
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Source: Internet
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Author: User
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Fluorescence recovery after photobleaching (FRAP) is a microscopy-based technique that can be used to ask how lipid raftsimpact protein and lipid diffusion in cells. This chapter, describes how to perform FRAP measurements of putative raft andnonraft proteins and lipids using a confocal microscope. Methods have been outlined for (1) transfecting cells with plasmidsencoding for the expression of green fluorescent protein-tagged proteins, (2) labeling cells with fluorescent lipid analogsor with the lipid-binding toxin cholera toxin B-subunit, (3) depleting and loading cholesterol into cell membranes using methyl-β-cyclodextrin,and (4) performing and analyzing confocal FRAP measurements.
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