title: A single step protein assay that is both detergent and reducer compatible: The cydex blue assay.
abstract: Determination of protein concentration is often an absolute prerequisite 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. It was 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, a single-step assay that is both detergent and reducer compatible was developed. 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.
authors.affiliation: Laboratory of Chemistry and Biology of Metals, CNRS UMR 5249 Grenoble, France. thierry.rabilloud@cea.fr.|Laboratory of Chemistry and Biology of Metals, Univ. Grenoble Alpes, Grenoble, France. thierry.rabilloud@cea.fr.|CEA Grenoble, BIG/CBM, Laboratory of Chemistry and Biology of Metals, CEA Grenoble, Grenoble, France. thierry.rabilloud@cea.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Rabilloud
mesh_terms: D000818:Animals; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003505:Cyclodextrins / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry*; D051379:Mice; D011506:Proteins / Q000032:analysis*; D000067996:RAW 264.7 Cells; D012394:Rosaniline Dyes / Q000737:chemistry*; D012710:Serum Albumin, Bovine
chemical_list: D003505:Cyclodextrins; D003902:Detergents; D007202:Indicators and Reagents; D011506:Proteins; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; C048139:Coomassie blue
keywords: Cyclodextrin; Detergents; Dye binding; Protein assay; RIPA buffer