Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.
In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.
Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center, Beijing 100850, China.
Yonghong
Y
Wu
Min
M
Wu
Guowei
G
He
Xiao
X
Zhang
Weiguang
W
Li
Yan
Y
Gao
Zhihui
Z
Li
Zhaoyan
Z
Wang
Chenggang
C
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000187:drug effects / Q000201:enzymology; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells / Q000187:drug effects; D010269:Paraquat / Q000737:chemistry / Q000494:pharmacology; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D055550:Protein Stability; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013447:Sulfites / Q000737:chemistry / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D029742:Caenorhabditis elegans Proteins; D029968:Escherichia coli Proteins; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; D013447:Sulfites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010269:Paraquat; C025026:sodium sulfite