A single step protein assay that is both detergent and reducer compatible: The cydex blue assay

TL;DR

This paper introduces the Cydex Blue assay, a single-step protein quantification method compatible with detergents and reducers, simplifying sample preparation for proteomics.

Contribution

The authors developed a novel Coomassie blue-based assay using cyclodextrins to enable detergent and reducer compatibility in protein measurement.

Findings

Compatible with up to 2% detergents and 5% mercaptoethanol or 50 mM DTT

Detects as low as 0.1 mg/mL protein in a single step

Uses cyclodextrins to complex detergents, ensuring assay resistance to reducers.

Abstract

Determination of protein concentration in often an absolute pre-requisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. We found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, we have thus developed a single step assay that is both detergent and reducer compatible. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5 % mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay. This article is protected by copyright. All rights reserved.