Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins

Abstract

The protein assay method of I. Polacheck and E. Cabib ( Anal. Biochem. 117, 311–314, 1981 ) has been modified to provide a general method for quantitating protein samples in the presence of detergents. Dilute detergent-solubilized membrane proteins, by using ribonucleic acid as a carrier, have been efficiently precipitated here by trichloroacetic acid (TCA) in the presence of sodium dodecyl sulfate (SDS). Washing the pellets once with TCA solution has removed most of the reagents present in the original sample. The washed sample could then be quantitated by the Lowry method ( O. J. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265–275, 1951 ). This procedure could be used to assay protein solutions of a concentration as low as 5 μg/ml in the presence of the following reagents: Triton X-100, Triton X-114, Tween 20, N-octylglucoside, deoxycholate, cholate, Thesit, octanoyl- N-methylglucamide, isotridecylpoly (ethyleneglycoether) n , Nonidet P-40, glucose, methyl- d-glucopyranoside, methyl- d-mannopyranoside, N-acetyl-glucosamine, Mn 2+, Ca 2+, Mg 2+, and many buffer reagents. Proteins solubilized from porcine brain myelin sheath and synaptic plasma membranes were quantitated by amino acid analysis and by the TCA SDS precipitation method described here. The resultant protein concentrations were almost identical. The results have suggested this TCA SDS precipitation method to be useful for quantitating dilute protein samples containing high concentrations of detergents and other reagents commonly employed in studying membrane proteins.