Elimination of interfering substances in the presence of detergent in the bicinchoninic acid protein assay.
Substantial time and effort are necessary to eliminate interference due to thiols and reducing sugars in the bicinchoninic acid protein assay when detergents such as sodium dodecyl sulfate (SDS) are present. A method to eliminate these interfering substances based on the binding of proteins to positively charged nylon membranes at pH 8.5 followed by methanol and aqueous washes is described. Color development using bicinchoninic acid occurs in solution with only a minor contribution from the nylon membrane itself. The retention of most proteins on the membrane is nearly quantitative and is only slightly decreased by high salt or detergent in the assay mixture. The interference due to thiol compounds such as beta-mercaptoethanol and dithiothreitol is completely eliminated while that due to reducing sugars such as glucose is substantially reduced. Therefore, the amount of protein dissolved in the sample buffer used for SDS-polyacrylamide gel electrophoresis (2% SDS and 5% beta-mercaptoethanol) can be determined with this bound protein assay.
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Department of Veterans Affairs, Medical Center, Nashville, Tennessee 37212.
D002623:Chemistry Techniques, Analytical / Q000379:methods; D003902:Detergents; D005069:Evaluation Studies as Topic; D008567:Membranes, Artificial; D009757:Nylons; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D011804:Quinolines
D003902:Detergents; D008567:Membranes, Artificial; D009757:Nylons; D011506:Proteins; D011804:Quinolines; C047117:bicinchoninic acid