QUANTITATION OF THE WATER CHANNEL PROTEIN AQUAPORIN (CHIP28) FROM RED-BLOOD-CELL MEMBRANES BY DENSITOMETRY OF SILVER-STAINED POLYACRYLAMIDEGELS

A protein determination procedure which involves the densitometry of s ilver stained polyacrylamide gels is described. It involves calibratio n with bovine serum albumin and molecular weight markers on the same g el 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 qu antitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It av oids interference by detergents and other substances usually present i n solutions of purified proteins. The procedure has been applied to th e quantitation of a recently identified protein, aquaporin (CHIP28), a ssumed to be a major water channel in the red blood cell membrane. A q uantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the prote in content of the sample, with the remainder comprising a glycosylated , high molecular mass component. The procedure may be useful for quant itating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.