Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels.
A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.
Benga
G
G
Department of Cell and Molecular Biology, University of Medicine and Pharmacy, Iuliu Hatieganu, Cluj-Napoca, Romania.
Banner
M
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Wrigglesworth
J M
JM
D000180:Acrylic Resins; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002462:Cell Membrane / Q000378:metabolism; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D016622:Silver Staining; D014882:Water-Electrolyte Balance
C496020:AQP1 protein, human; D000180:Acrylic Resins; D020346:Aquaporins; D001789:Blood Group Antigens; D007473:Ion Channels; D008565:Membrane Proteins; C016680:polyacrylamide gels; D051398:Aquaporin 1