Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE.
Measuring the protein content of a sample is a mandatory and frequently practiced procedure in the lab. Although the procedure is quite simple and convenient to perform with commercially available kits, incompatible reagents in the lysate can cause problems in the quality of measurement. Unfortunately these reagents are cornerstones of high efficiency lysing buffers, e.g. high amounts of urea or beta-mercaptoethanol. In this study we addressed the tolerance of the well-known BCA-assay (bicinchoninic acid) to various reagents in different concentrations, with special regard to a subsequent 2D-gelelectrophoresis. As a result, the kit is incompatible with the recipes of regular 2D-buffers. Also, when mixing two different reagents interfering effects will occur in a non-predictable way. Therefore we established a new method to quantify protein content in lysates ready for 2D-gelelectrophoresis: by mixing an aliquot with SDS, an equilibration is performed to that the sample can be run on a regular 1D SDS PAGE. Image analysis following fluorescence staining (SYPRO Ruby) reveals the absolute protein content in comparison to a BSA dilution curve processed accordingly.
Krieg
Rene C
RC
Institute of Pathology, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany. rkrieg@ukaachen.de
Dong
Yan
Y
Schwamborn
Kristina
K
Knuechel
Ruth
R
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D011804:Quinolines / Q000737:chemistry*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011506:Proteins; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; C024795:bicinchoninate reagent