Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments.
To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.
Kao
Shao-Hsuan
SH
Institute of Biochemistry and Biotechnology, College of Medicine, Chung-Shan Medical University, Taichung, Taiwan.
Wong
Hin-Kwan
HK
Chiang
Chia-Ying
CY
Chen
Han-Min
HM
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003124:Colorimetry / Q000295:instrumentation* / Q000379:methods; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D011804:Quinolines / Q000032:analysis; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D011506:Proteins; D011804:Quinolines; D012710:Serum Albumin, Bovine; D014508:Urea; C047117:bicinchoninic acid; D004229:Dithiothreitol