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Brca1

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BRCA1 is a laboratory equipment product developed by Merck Group. It is a tool used for the analysis and detection of the BRCA1 gene, which is associated with an increased risk of certain types of cancer. The core function of BRCA1 is to facilitate the genetic testing and screening of individuals for mutations in the BRCA1 gene.

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Lab products found in correlation

γh2ax, by Merck Group (5 mentions) Fancd2, by Novus Biologicals (2 mentions) Phosphorylated h2ax γh2ax, by Merck Group (2 mentions) Rad51, by Novus Biologicals (2 mentions) Chemiluminescence, by Cytiva (1 mentions) Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions) Rad51, by Merck Group (1 mentions) Axiovision camera and software, by Olympus (1 mentions) Prolong gold anti fade reagent containing dapi, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Fluorescent microscope, by Olympus (1 mentions) Dynll1, by Abcam (1 mentions) Rad50, by Cell Signaling Technology (1 mentions) Mre11, by GeneTex (1 mentions) α and β tubulin, by Merck Group (1 mentions) Atmin, by Merck Group (1 mentions) Parp1, by Cell Signaling Technology (1 mentions) Flag m2, by Merck Group (1 mentions) Histone h3, by Abcam (1 mentions) Tubulin, by Cell Signaling Technology (1 mentions)

13 protocols using brca1

1

Antibody Detection and Characterization

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Several commercially available primary antibodies were utilized: RAD51D (Novus), XRCC2 (Santa Cruz), BRCA1 (Millipore) and BRCA2 (Calbiochem). Anti-RAD51C, anti-XRCC3, anti-γH2AX, anti-HA, and anti-β actin antibodies were as described elsewhere.[24 (link)]
Secondary antibodies for immunofluorescence microscopy and the detection of immunoblots using chemiluminescence (Amersham) were as described previously.[23 (link)]
Park J.Y., Virts E.L., Jankowska A., Wiek C., Othman M., Chakraborty S.C., Vance G.H., Alkuraya F.S., Hanenberg H, & Andreassen P.R. (2016). COMPLEMENTATION OF HYPERSENSITIVITY TO DNA INTERSTRAND CROSSLINKING AGENTS DEMONSTRATES THAT XRCC2 IS A FANCONI ANEMIA GENE. Journal of medical genetics, 53(10), 672-680.
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2

Isolation and Characterization of HPV-Positive Keratinocytes

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Healthy human foreskin keratinocytes (HFKs) were isolated from deidentified neonatal specimens. HFK cells were transfected with HPV16 or HPV31 genomes to generate HFK-16 and HFK-31 cell lines that contain episomal HPV DNA as previously described (18 (link)). CIN 612 cells were isolated from a cervical cancer biopsy specimen and contain HPV31 episomes (19 (link)). All keratinocytes were cocultured in E-media with growth-arrested NIH 3T3 fibroblasts as described previously (3 (link)). Antibodies to phosphorylated CHK1 (pCHK1) (catalog no. 12302; Cell Signaling Technology [CST]), phosphorylated CHK2 (pCHK2) (catalog no. 2661; CST), FANCD2 (catalog no. 100182; Novus), phosphorylated H2Ax (γH2AX) (catalog no. 05636; Millipore), phosphorylated SMC1 (pSMC1) (catalog no. 4805S; CST), BRCA1 (catalog no. OP92; Millipore), RAD51 (catalog no. NB100148; Novus), and TOP2β (catalog no.A300-950A; Bethyl Laboratories) were used.
Kaminski P., Hong S., Kono T., Hoover P, & Laimins L. (2021). Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication. mBio, 12(1), e00005-21.
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3

DNA Damage Response Foci Assay

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The γ-H2AX, 53BP1, RAD51, and BRCA1 (Millipore, Billerica, MA, USA; Santa Cruz Biotechnology, Santa Cruz Biotechnology, Santa Cruz, CA, USA and Novus Biologics, Littleton, CO, USA, respectively) IR-induced foci assay was performed as previously described.43 (link) For γ-H2AX and 53BP1 foci, cells were treated with 2 Gy and collected at 1, 4, 8, and 24 h. Cells were collected at 4, 8, and 24 h post-12 Gy for assessment of BRCA1 foci. RAD51 foci were assessed 8 h post-IR, as previously described.43 (link) Cells were plated on coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 (Sigma-Aldrich), blocked in 10% FBS, and incubated with primary antibodies (1 : 300) for 1 h at room temperature. The coverslips were washed, blocked with 10% FBS, and incubated with a AlexaFluor-488 secondary antibody (1 : 400, Invitrogen) for 45 min at room temperature. Coverslips were washed a final time and mounted on slides using ProlongGold anti-fade reagent containing DAPI (Applied Biosystems, Grand Island, NY, USA). Foci were imaged using an Olympus fluorescent microscope equipped with an AxioVision camera and software. Cells were scored as positive if they contained four or more foci/nuclei and results are presented as percent positive cells (% Pos).
Martin N.T., Nakamura K., Paila U., Woo J., Brown C., Wright J.A., Teraoka S.N., Haghayegh S., McCurdy D., Schneider M., Hu H., Quinlan A.R., Gatti R.A, & Concannon P. (2014). Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks. Cell Death & Disease, 5(3), e1130-.
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4

Comprehensive Antibody Panel for DNA Repair

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Mouse antibodies used were against Flag M2, α- and β-tubulin (Sigma), BRCA1, γH2AX (Millipore), LIG4, GFP and c-myc (Santa Cruz), REV7 (BD Transduction Libratory); rabbit antibodies were against PARP1, H2AX, FEN1, RAD50 (Cell Signalling), Histone H3, BLM, DNA2, DYNLL1 (Abcam), MRE11 (GeneTex), ATMIN (Millipore), EXO1 (Sigma), PTIP, 53BP1and Rad51 (Santa Cruz), RPA32 Phosphor-S4/S8 (Bethyl); goat antibodies were against NBN (Santa Cruz) and rat antibody against RPA2 (Cell Signaling).
He Y.J., Meghani K., Caron M.C., Yang C., Ronato D.A., Bian J., Sharma A., Moore J., Niraj J., Detappe A., Doench J.G., Legube G., Root D.E., D’Andrea A.D., Drané P., De S., Konstantinopoulos P., Masson J.Y, & Chowdhury D. (2018). DYNLL1 binds MRE11 to limit DNA end resection in BRCA1-deficient cells. Nature, 563(7732), 522-526.
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5

Antibody Panel for DNA Repair Pathway

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Primary antibodies used for Western blots included BRCA1 (Santa Cruz Biotechnology), BRCA1 (Millipore), BRCA2 (Millipore), 53BP1 (Cell Signaling), RIF1 (Bethyl Laboratories), REV7 (BD Transduction Laboratories), PTIP (kindly provided by Dr. Junjie Chen, MD Anderson Cancer Center), KU70 (GeneTex), Tubulin (Cell Signaling), and GAPDH (EMD Millipore).
Yazinski S.A., Comaills V., Buisson R., Genois M.M., Nguyen H.D., Ho C.K., Todorova Kwan T., Morris R., Lauffer S., Nussenzweig A., Ramaswamy S., Benes C.H., Haber D.A., Maheswaran S., Birrer M.J, & Zou L. (2017). ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes & Development, 31(3), 318-332.
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6

DNA Damage Response Signaling Pathway

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p16 (#550834; BDPharmingen), phosphorylated CHK1 (pCHK1) (#12302; CST), phosphorylated CHK2 (pCHK2) (#2661; CST), FANCD2 (#100182; Novus), phosphorylated H2Ax (γH2AX) (#05636 Millipore), phosphorylated SMC1 (pSMC1) (#4805S; CST), BRCA1 (#OP92; Millipore), RAD51 (#NB100148; Novus), APOBEC3A (#HPA043237; Sigma), APOBEC3B (#NBP256411; Novus), APOBEC3B (mAb 5210-87-13) (Gift from Reuben Harris lab)) [31 (link)]
Kono T., Hoover P., Poropatich K., Paunesku T., Mittal B.B., Samant S, & Laimins L.A. (2020). Activation of DNA damage repair factors in HPV positive oropharyngeal cancers.. Virology, 547, 27-34.
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7

Profiling DNA Damage Response Proteins

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The commercial antibodies used were as follows: ATM (clone MAT3-4G10/8; Sigma-Aldrich, A1106), Rad52 (Cell Signaling Technology, #3425), Ku80 (Cell Signaling Technology, #2180), BRCA1 (Millipore, #07-434), γH2AX (Millipore, #05-636; Cell Signaling Technology, #9718), 53BP1(Bethyl Laboratories, A300-272A), XRCC4 (Novus, NBP1-30878), Rad51 (Thermo Scientific, MA1-23271), DNA-PK (Thermo Scientific, MA5-15813), phospho-ATM S1981 (Thermo Scientific, MA1-2020), V5 (Serotec, MCA1360), BrdU (Roche, 11170376001), Cas9 mAb (Diagenode, C15200203), RPA2 (Millipore, #MABE285), and phospho-RPA2-S4/S8 (Bethyl Laboratories, A300-245A). Fibrillarin antibody (mAb clone72B9) was a gift from U. Scheer (Wurzburg). Antibodies against UBF, Treacle, Nop52, Rrn3 and Paf49 were raised in sheep against recombinant proteins. For double-labeling experiments, 53BP1 and BRCA1 antibodies were directly labeled using Zenon tricolor rabbit IgG-labeling kit #2 (Life Technologies) according to the manufacturer's instructions. Secondary antibodies for immunofluorescence were purchased from Jackson ImmunoResearch.
van Sluis M, & McStay B. (2015). A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes & Development, 29(11), 1151-1163.
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8

Western Blot Analysis of Breast Cancer Biomarkers

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Cells were collected for western blot analysis as described.46 (link) Protein concentration was determined by BCA protein assay (Bio-Rad, Hemel Hempstead, UK). Twenty micrograms of protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the following antibodies at 4 °C for overnight: BRCA1 (1:1000, Millipore; 07-434, Watford, UK)23 (link), EZH2 (1:1000, Diagenode, Seraing/Ougrée, Belgium; C15410039), ERα (1:1000, Santa-Cruz, Insight Biotechnology Ltd, Wembley, UK; sc-7207), β-tubulin (1:1000, Santa-Cruz); FOXO3 (1:3000, Millipore) and GATA3 (1:1000, Santa-Cruz; H-48). On the second day, the membranes were washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibody (1:30000, DAKO, Ely, UK) for 1 h. The chemilluminance signals were detected by incubating the membranes with ECL substrate (Perkin Elmer, Seer Green, UK) and exposed to X-ray films (GE Healthcare, Amersham, UK).
Gong C., Yao S., Gomes A.R., Man E.P., Lee H.J., Gong G., Chang S., Kim S.B., Fujino K., Kim S.W., Park S.K., Lee J.W., Lee M.H., Khoo U.S, & Lam E.W. (2016). BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer. Oncogenesis, 5(4), e214-.
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9

Immunoprecipitation and Immunoblotting Analysis

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For immunoprecipitation, whole-cell extracts from cells were resuspended in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mMNaCl, 1% Nonidet P-40, 5% glycerol, 1 mM EDTA, 1 mM MgCl2, 1 mM ATP, 1 mM DTT, 10 mM NaF, 1 mM sodium vanadate, and protease inhibitors. One milligram of total cell extract was incubated with the indicated antibodies and immunoprecipitated with protein G-conjugated agarose beads (GE Healthcare). For immunoblotting, ∼30–50 μg of protein was subjected to SDS-PAGE analysis. Antibodies against each protein were purchased from the following companies: CHK1, Cyclin A, Cyclin E, CUL1, and CAF1 from Santa Cruz Biotechnology; p-CHK1(Ser345), ASF1a, ASF1b, and βTRCP from Cell Signaling Technology; ATR from Affinity Bioreagent; ATM from Novus Biological; and BRCA1 from Milipore.
Im J.S., Keaton M., Lee K.Y., Kumar P., Park J, & Dutta A. (2014). ATR checkpoint kinase and CRL1βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks. Genes & Development, 28(8), 875-887.
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10

Multimodal Analysis of DNA Damage Signaling

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The following antibodies were used: ATR (sc1887 N19); RAD51 (sc8349); RPA (Cell Signaling Technology, Danvers, MA, USA, 4E4); P-ATM (sc47739); H2B (ab52484); H2BK120Ub (Cell Signaling Technology, D11); H3 (sc10809); Actin (ab6276); CDC73 (Cell Signaling Technology, D38E12); P-RPA (Bethyl Laboratories NB100-S44); P-ATR (Cell Signaling Technology, S428); P-Chk1 (Cell Signaling Technology, 133D3); Chk1 (Cell Signaling Technology, 261DS); 53BP1 (ab36823); GFP (sc8334); PARP1 (sc7007 F2); KU70 (sc17789 E5); CtIP (Bethyl Laboratories, Inc., Montgomery, TX, USA,A300-488A); BRCA1 (sc642); γH2AX (Millipore, Merck Chemicals and Life Science AB, Solna, Sweden, JBW301); PAF1 (ab137519); GAPDH (sc365062); KU86 (sc528); Fibrillarin (ab5821); UBA1 (Cell Signaling, 4890); CAND1 (Bethyl Laboratories, Inc. A302-901A-T); RNF20 (Cell Signaling Technology, 9425); RNF40 (Bethyl Laboratories, A300-719A-T); RUVBL2 (Bethyl Laboratories, A302-536A-T); CUL1 (Bethyl Laboratories, A303-373A-T); FBXO21 (ab179818); Flour-555 (A21434, Invitrogen, Life Technologies Europe BV, Stockholm, Sweden); Alexa Flour-488; Phalloidin 594 (Sigma-Aldrich Company Ltd, Dorset, UK, 51927); mouse, rabbit, rat horseradish peroxidase-conjugated ab (Abcam, Cambridge, UK); mouse and rabbit IRDye-conjugated ab (Licor, Lincoln, NE, USA).
Herr P., Lundin C., Evers B., Ebner D., Bauerschmidt C., Kingham G., Palmai-Pallag T., Mortusewicz O., Frings O., Sonnhammer E, & Helleday T. (2015). A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair. Cell Discovery, 1, 15034-.
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