Evaluation of different commercial antibodies for their ability to detect human and mouse tissue factor by western blotting

Abstract

Background: Western blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. Objectives: We assessed several commercial anti-human and anti-mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. Material and Methods: We used human pancreatic cancer cell lines expressing different levels of TF and a mouse pancreatic cancer cell line expressing TF with a matched knockout derivative. Results: Human and mouse TF protein detected by western blotting correlated with levels of TF mRNA in these cell lines. The apparent molecular weight of TF is increased by N-linked glycosylation and, as expected, deglycosylation decreased the size of TF based on western blotting. We found that four commercial anti-human TF antibodies detected TF in a TF-positive cell line HPAF-II whereas no signal was observed in a TF-negative cell line MIA PaCa-2. More variability was observed in detecting mouse TF. Two anti-mouse TF antibodies detected mouse TF in a TF-positive cell line and no signal was observed in a TF knockout cell line. However, a third anti-mouse TF antibody detected a nonspecific protein in both the mouse TF-positive and TF-negative cell lines. Two anti-human TF antibodies that are claimed to cross react with mouse TF either recognized a nonspecific band or did not detect mouse TF. Discussion: Our results indicate that there is a range in quality of commercial anti-TF antibodies. Conclusion: We recommend that all commercial antibodies should be validated to ensure that they detect TF.