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

Abstract

BackgroundWestern blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. ObjectivesWe assessed several commercial anti‐human and anti‐mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. Material and MethodsWe 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. ResultsHuman 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. DiscussionOur results indicate that there is a range in quality of commercial anti‐TF antibodies. ConclusionWe recommend that all commercial antibodies should be validated to ensure that they detect TF.