A double-label two-dimensional procedure for the analysis of membrane proteins.
A previously described double-label two-dimensional electrophoresis procedure (Wheeler et al., Anal. Biochem. 1986 159, 1-7) for the analysis of differences between two complex mixtures of soluble proteins has been modified to allow analysis of proteins requiring detergent for aqueous solubility. The samples are first disrupted by sonication and the insoluble proteins concentrated by high-speed centrifugation. The proteins are then solubilized with sodium dodecyl sulfate and further concentrated in a centrifugal concentrator to achieve protein mixtures suitable for labeling with 14C and 3H by reductive methylation and subsequent two-dimensional electrophoresis. The sample concentration step is quick, minimizes the concentration of sodium dodecyl sulfate in the final sample, and avoids the potential difficulties associated with lyophilization or precipitation. The modified procedure was applied to the analysis of erythrocyte membranes, platelets and isolated placental microvilli. The high resolving power of two-dimensional gel electrophoresis is retained and the procedure is sensitive because the conditions of labeling allow substantial incorporation of radioactivity into protein despite the presence of detergent.
Wheeler
T T
TT
Biochemistry Department, Victoria University of Wellington, New Zealand.
Jordan
T W
TW
Ford
H C
HC
D001792:Blood Platelets / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D008871:Microvilli / Q000032:analysis; D010920:Placenta / Q000032:analysis / Q000648:ultrastructure; D011247:Pregnancy; D011480:Protease Inhibitors; D015203:Reproducibility of Results
D008565:Membrane Proteins; D011480:Protease Inhibitors