{"doi":"10.1016/j.rpth.2025.103016","title":"Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells","abstract":"Background: The tissue factor (TF)/activated factor VII complex is the major activator of the coagulation system. TF is expressed by a variety of cells, including activated monocytes and tumor cells. Increased TF expression can cause thrombosis in different diseases, including sepsis, viral infections, and cancer. We have previously described a method for analyzing human TF in high-expressing cells by Western blotting. Objectives: The goal of this study was to establish a method for detecting human TF in low-expressing cells. Methods: We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam). We also used the Abcam antibody to measure TF expression in lipopolysaccharide-stimulated peripheral blood mononuclear cells. Results: We found that sensitivity was affected by various factors, including the blocking conditions, the detection method, and the primary and secondary antibodies. Both the R&D and Abcam antibodies were more specific in assessing TF expression than the Novus antibody; however, the Abcam antibody was the best of the 3 in evaluating TF in low-expressing cell lines. We detected TF in lipopolysaccharide-stimulated human peripheral blood mononuclear cells using the new method with the Abcam antibody. Conclusion: Researchers should consider each step in Western blotting when establishing a method for detecting low-abundance antigens, such as TF.","journal":"Research and Practice in Thrombosis and Haemostasis","year":2025,"id":534492,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9613,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":864650,"name":"Ana Teresa Azevedo Sachetto","orcid":"0000-0002-8234-1209","position":1,"is_corresponding":false},{"id":243111,"name":"Nigel Mackman","orcid":"0000-0002-9170-7700","position":2,"is_corresponding":false},{"id":1417641,"name":"Megan V. Perkins","orcid":null,"position":0,"is_corresponding":true}],"reference_count":40,"raw_metadata":null,"created_at":"2026-07-19T02:51:47.434742Z","pmid":"40994889","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}