title: An endogenous 'non-specific' protein detected by a His-tag antibody is human transcription regulator YY1.
abstract: Histidine-tags have been used for a wide variety of experiments including protein purification, Western blots, immunoprecipitation and immunohistochemistry. In our previous studies, we have repeatedly detected a 'non-specific' endogenous protein of about 60 kD in Western blots of protein lysates from HEK293T or HeLa cells using the anti-His-tag antibody (His-probe (H3), catalogue #, SC-8036, Santa Cruz Biotech. Co.) (Yu et al., J. Biol. Chem. 284 (2009) 1505-1513). Here we have immunoprecipitated the protein from HeLa nuclear extracts using the anti-His-tag antibody, excised the 60 kD band and subjected it to LC-MS/MS (Fig. 1). The deduced sequences of two peptides of the protein match the human transcriptional regulator YY1 (Yin and Yang 1, UniProt ID, P25490, Fig. 2), which contains 11 histidine residues in a stretch (from amino acid 70 to 80) at its NH2-terminal region without known functions (Lee et al., Nucleic Acids Res. 23 (1995) 925-931; Bushmeyer et al., J. Biol. Chem. 270 (1995) 30213-30220). Since genes encoding other Histidine-repeat proteins also exist in the genome (Salichs et al., PLoS Genet. 5 (2009) e1000397), it is possible that YY1 might not be the only endogenous protein that could be expressed and recognized by the antibody in different sources of samples in future experiments. The presence of various endogenous histidine-repeat proteins suggests that data from experiments particularly immunostaining using His-tag antibodies need to be interpreted with caution. This might also be useful to the broader scientific community by providing an example for the interpretation of 'non-specific' bands in Western blots.
authors.affiliation: Department of Biochemistry & Medical Genetics, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada R3E 0J9.
authors.forename: Niaz
authors.initials: N
authors.lastname: Mahmood
authors.affiliation: Department of Biochemistry & Medical Genetics, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada R3E 0J9 ; Physiology & Pathophysiology, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada R3E 0J9.
authors.forename: Jiuyong
authors.initials: J
authors.lastname: Xie
keywords: HEK293T; HeLa; His-tag antibody; Histidine-rich proteins; Yin and Yang 1 (YY1); ‘Non-specific’ band