Many antibodies became commercially available without careful characterization or proper quality control analysis. overall performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels. Keywords:BRCA1, Antibody validation, Western blot, Immunoprecipitation, Chromatin Immunoprecipitation, Immunofluorescence == Introduction == Antibodies (Abs) are a important resource and one of most frequently used tools in biomedical research1,2. You will find more than one million commercially available antibodies on offer1, however, demanding antibody validation efforts tend to fall behind antibody generation. Utilization of poor quality antibodies has been estimated to cost $350 million in the United States and $800 million globally each year as a result of unreliable experiments3. Ansatrienin B Germline mutations in tumor suppressor geneBRCA1are highly penetrant for the increased risk of familial breast and ovarian malignancy occurrence4-6. A wealth of molecular functions of BRCA1 has been recognized since the gene was recognized in 1994. Human BRCA1 is a large protein with multiple functional domains and forms several unique complexes that are involved in many important cellular activities such as DNA damage repair, cell cycle checkpoint control, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation7-15. Over the years, a large number of BRCA1 antibodies have been generated and become commercially available. It is relatively common in the BRCA1 field that many antibodies were in the beginning confirmed based on simple Western blotting analysis, but eventually used in numerous applications without demanding antibody characterization. For example, published BRCA1 ChIP and ChIP-seq results in literature rarely overlapped or are hard to reproduce16,17. Although this inconsistency could be attributed to difference in cell lines or other potential experimental contextual differences, it is equally possible that many BRCA1 antibodies used in studies recognize strong nonspecific bands on Western blotting, or their specificity is poorly characterized, therefore could introduce high noise background in ChIP. The problem is hard to tackle because BRCA1 is not known to bind to DNA in a sequence-specific manner and could bind anywhere through protein-protein interactions. Currently there are no simple alternative ways to verify BRCA1 target regions identified by ChIP analysis, making it especially important that BRCA1 antibodies are truly specific. Of note, most of our current knowledge of BRCA1 derived Ansatrienin B from studies using human cancer cell lines. Studies using BRCA1 mouse models have not been fully explored, especially at molecular level using mouse derived cells. For example, after 30 years of extensive studies, the role of mouse BRCA1 and even its expression pattern in cell and tissue types are still unclear. This is partly due to lacking well characterized antibodies recognizing mouse BRCA1. Investigators sometimes performed Western blotting with BRCA1 antibodies that are unverified or no detailed information provided18-20, or by using genomic DNA and/or mRNA analyses as indicative of BRCA1 deletion/depletion in transgenic animal studies21-25. In our studies of BRCA1 using mouse cells, we found that several commercial BRCA1 NIK antibodies did recognize a band with expected size on Western blot, however, the intensity of the presumed BRCA1 band did not change when endogenous BRCA1 was knocked down using siRNA. While we confirmed the knockdown of Ansatrienin B Ansatrienin B BRCA1 at mRNA level by RT-PCR, a caveat of this approach is that we could not rule out the possibility that BRCA1 mRNA and protein level may not always be in sync. We therefore decided to carry out a thorough rigorous characterization of all commercial BRCA1 antibodies in terms of their specificity in human and mouse cells in several common applications. The International Working Group on Antibody Validation (IWGAV) in 2016 proposed five conceptual pillars to guide antibody validation in specific research applications: (1) genetic strategies: measure the relevant signal in control cells or tissues in which the target gene has been knocked down or knocked out (KO); (2) orthogonal strategies: use an antibody-independent method for quantification across multitudes of samples and then examine the correlation between the antibody-based and antibody-independent quantifications; (3) independent antibody strategies: use two or more independent antibodies that recognize different epitopes on the target protein and.