ACTB and GAPDH proteins appear at multiple positions of SDS-PAGE and may not be suitable for serving as reference genes for the protein level determination in such techniques as Western blotting

Most human genes can produce multiple protein isoforms that should appear at multiple positions of polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS), but most published results of Western blotting show only one protein. We performed SDS-PAGE of proteins from several human cell lines, isolated the proteins at the 72-, 55-, 48-, 40-, and 26-kD positions, and used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although ACTB and GAPDH are 41.7-kD and 36-kD proteins, respectively, LC-MS/MS identified peptides of ACTB and GAPDH at all of these SDS-PAGE positions, making us wonder whether they produce some unknown protein isoforms. The NCBI (National Center for Biotechnology Information, USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one non-coding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatic analysis identified a 17.6-kD isoform encoded by the non-coding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with great similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to ACTB. These data collectively disqualify GAPDH and ACTB from serving as the reference genes for determination of the protein level in such techniques as Western blotting, a leading role these two genes have been playing for decades in the biomedical research.