Analysis of the association of proteins with membranes.
Proteins associate with membranes in a number of ways. Is the membrane an integral membrane protein? A peripheral protein? Does it have a glycosylphosphoinositol (GPI) tail? Is it associated with caveolae-derived plasma membrane domains? This unit describes methods for determining how a protein of interest associates with the membrane including extraction with alkaline carbonate, urea, high salt, or Triton X-114. GPI linkage is probed using PI-PLC cleavage. There is a method for detergent solubilization of Triton-X 100 insoluble integral and GPI-linked proteins. Caveolae membranes can be isolated using Triton X-100 or a detergent-free method.
Okamoto
T
T
Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Schwab
R B
RB
Scherer
P E
PE
Lisanti
M P
MP
D000818:Animals; D021941:Caveolae / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D017830:Octoxynol; D009994:Osmolar Concentration; D054801:Phosphoinositide Phospholipase C; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D011480:Protease Inhibitors; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012965:Sodium Chloride; D014508:Urea
D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011480:Protease Inhibitors; D011506:Proteins; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D011189:Potassium Chloride; D014508:Urea; D017830:Octoxynol; C010615:Nonidet P-40; D054801:Phosphoinositide Phospholipase C