ACTB and GAPDH appear at multiple SDS-PAGE positions, thus not suitable as reference genes for determining protein loading in techniques like Western blotting.

ABSTRACT: We performed polyacrylamide gel electrophoresis of human proteins with sodium dodecyl sulfate, isolated proteins at multiple positions, and then used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 41.7 and 36 kDa proteins, respectively, LC-MS/MS identified their peptides at all the positions studied. The National Center for Biotechnology Information (USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one noncoding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatics analysis identified a 17.6 kDa isoform encoded by the noncoding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but so

SUBMITTER: Zhang K 

PROVIDER: S-EPMC8669867 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature