Evaluation of different commercial antibodies for their ability to detect human and mouse tissue factor by western blotting.
antibody
glycosylation
tissue factor
validation
western blotting
Journal
Research and practice in thrombosis and haemostasis
ISSN: 2475-0379
Titre abrégé: Res Pract Thromb Haemost
Pays: United States
ID NLM: 101703775
Informations de publication
Date de publication:
Aug 2020
Aug 2020
Historique:
received:
12
03
2020
revised:
11
04
2020
accepted:
16
04
2020
entrez:
1
9
2020
pubmed:
31
8
2020
medline:
31
8
2020
Statut:
epublish
Résumé
Western blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. We assessed several commercial anti-human and anti-mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. 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. 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. Our results indicate that there is a range in quality of commercial anti-TF antibodies. We recommend that all commercial antibodies should be validated to ensure that they detect TF.
Sections du résumé
BACKGROUND
BACKGROUND
Western blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation.
OBJECTIVES
OBJECTIVE
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
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
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
CONCLUSIONS
Our results indicate that there is a range in quality of commercial anti-TF antibodies.
CONCLUSION
CONCLUSIONS
We recommend that all commercial antibodies should be validated to ensure that they detect TF.
Identifiants
pubmed: 32864552
doi: 10.1002/rth2.12363
pii: S2475-0379(22)02067-2
pmc: PMC7443430
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1013-1023Subventions
Organisme : NCI NIH HHS
ID : P30 CA016086
Pays : United States
Organisme : NCI NIH HHS
ID : R01 CA211098
Pays : United States
Organisme : NHLBI NIH HHS
ID : U01 HL143403
Pays : United States
Informations de copyright
© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.
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