Sedimentation equilibrium of detergent-solubilized membrane proteins in the preparative ultracentrifuge.
Working with detergent-solubilized bacteriorhodopsin we have used a table top preparative centrifuge for determination of M(r) of membrane proteins by sedimentation equilibrium. We demonstrate the use of two new methods to measure protein concentration as a function of distance from rotor axis: (i) peak integration after HPLC on silica gel, and (ii) microdensitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. These methods, although somewhat lengthier than conventional spectrophotometric methods, are more reliable, especially in the presence of a large amount of detergent and small amount of protein. In addition they provide independent information on the status of the protein after sedimentation equilibrium, the association of the solubilized units being readily detected by gel chromatography and proteolytic cleavage by SDS-PAGE.
Garrigos
M
M
Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Centeno
F
F
Deschamps
S
S
Møller
J V
JV
le Maire
M
M
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D019617:Halobacterium salinarum / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D012995:Solubility; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods*
D003902:Detergents; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D001436:Bacteriorhodopsins; D017830:Octoxynol