Isolation and use of rafts.
This unit describes methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft-like membranes isolated by other methods, they are referred to here as detergent-resistant membranes (DRMs). DRMs can be isolated by flotation on sucrose density gradients or by pelleting after detergent extraction. DRM proteins can be analyzed by SDS-PAGE and immunoblotting. Additionally, radiolabeled DRM proteins can be analyzed, and lipids can be quantitated by high-performance thin layer chromatography. Support protocols needed for the lipid analysis are also provided. Finally, protocols for raft disruption by cholesterol removal and measuring the kinetics of such removal are included together with a method that reverses the cholesterol removal (cholesterol repletion).
Brown
Deborah A
DA
State University of New York at Stony Brook, Stony Brook, New York.
D002498:Centrifugation; D002784:Cholesterol / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000737:chemistry; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D013395:Sucrose; D047392:beta-Cyclodextrins / Q000737:chemistry
D003902:Detergents; D008565:Membrane Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D013395:Sucrose; D002784:Cholesterol