The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

Sawicka, M., Pawlikowski, J., Wilson, S., Ferdinando, D., Wu, H., Adams, P.D., Gunn, D.A. and Parish, W. (2013) The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS ONE, 8(1), e53313. (doi: 10.1371/journal.pone.0053313) (PMID:23308192) (PMCID:PMC3540092)

Abstract

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Pawlikowski, Mr Jeff and Adams, Professor Peter
• Authors: Sawicka, M., Pawlikowski, J., Wilson, S., Ferdinando, D., Wu, H., Adams, P.D., Gunn, D.A., and Parish, W.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• Copyright Holders: Copyright © 2013 The Authors
• First Published: First published in PLoS ONE 8(1):e53313
• Publisher Policy: Reproduced under a Creative Commons License