Brca2

Manufactured by Fortis Life Sciences

BRCA2 is a gene that provides instructions for making a protein that plays a role in repairing damaged DNA. This gene is considered a tumor suppressor gene, as it helps prevent cells from growing and dividing too rapidly or in an uncontrolled way.

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Lab products found in correlation

Rad51, by Santa Cruz Biotechnology (3 mentions) Rad18, by Fortis Life Sciences (2 mentions) Rad51, by Abcam (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) γh2ax, by Merck Group (2 mentions) Tubulin, by Cell Signaling Technology (1 mentions) Palb2, by Fortis Life Sciences (1 mentions) Phospho chk1, by Cell Signaling Technology (1 mentions) Rnf168, by Merck Group (1 mentions) Pold3, by Fortis Life Sciences (1 mentions) Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) Flag tag (flag), by Cell Signaling Technology (1 mentions) Rucaparib, by Selleck Chemicals (1 mentions) Rabbit anti β actin, by Abcam (1 mentions) Pyrvinium pamoate, by Merck Group (1 mentions) Vinculin, by Cell Signaling Technology (1 mentions) Wnt c59, by Selleck Chemicals (1 mentions) Pri 724, by Selleck Chemicals (1 mentions) Mouse anti β actin, by Abcam (1 mentions) Anti rat hrp, by Jackson ImmunoResearch (1 mentions)

5 protocols using brca2

Western Blot Analysis of DNA Damage Response Proteins

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Nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) and whole cell extracts were generated using RIPA buffer with protease and phosphatase inhibitors added. Proteins were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gelelectrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline tween 20 (PBST) at room temperature for 1 h. Primary antibodies were incubated overnight at 4° and horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 1 h at room temperature. The following primary antibodies were used: BRCA1 (EMD Millipore, catalog# OP92), RNF168 (EMD Millipore, #06-1130-I), Tubulin (Cell Signaling, catalog# 2148), GFP (Santa Cruz Biotechnology, catalog# sc-9996), RFP (ChromoTek, catalog# 6g6-20), FLAG (Cell Signaling, catalog# 14793 and Sigma Aldrich, catalog# F1804), phospho-Chk1 (Cell Signaling, catalog# 2344), Chk1 (Cell Signaling, catalog# 2360), POLD3 (Bethyl Laboratories, A301-244A), PALB2 (Bethyl Laboratories, catalog# A301-246A), BRCA2 (Bethyl Laboratories, catalog# A303-435A), RAD51 (Santa Cruz Biotechnology, catalog# sc-8349), RAD18 (Bethyl Laboratories, catalog# A301-340A), 53BP1 (Cell Signaling, catalog# 4908).

Krais J.J, & Johnson N. (2020). Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks. Nucleic Acids Research, 48(8), 4298-4308.

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Comprehensive Biochemical Assay Protocol

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Olaparib, rucaparib, WNT-C59, and PRI724 were obtained from Selleckchem. Pyrvinium pamoate was obtained from Sigma Aldrich. The following antibodies were obtained from the indicated suppliers: BRCA2 (Bethyl, Cat#A303-434A, 1:2000), Vinculin (Cell Signaling Technology, Cat#13901, 1:1000), WNT3A (R&D systems, Cat# MAB9025-100, 1:1000), γH2Ax (Ser139) (EMD Millipore, Cat# 05-636, 1:1000), mouse anti-β-Actin (Abcam, Cat# ab6276, 1:10,000), Rabbit anti-β-Actin (Abcam, Cat# ab8227, 1:10,000), Rad51 (Abcam, Cat# ab176458). Anti-rat HRP (Jackson ImmunoResearch, Cat# 112-035-062, 1:5000)

Yamamoto T.M., McMellen A., Watson Z.L., Aguilera J., Ferguson R., Nurmemmedov E., Thakar T., Moldovan G.L., Kim H., Cittelly D.M., Joglar A.M., Brennecke E.P., Wilson H., Behbakht K., Sikora M.J, & Bitler B.G. (2019). Activation of Wnt signaling promotes olaparib resistant ovarian cancer. Molecular carcinogenesis, 58(10), 1770-1782.

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Characterization of DNA Repair in Cancer Cell Lines

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H1299 (human non-small cell lung carcinoma) and A2780 (human ovarian cancer) were obtained from ATCC, and HCT116 and HCT116 RAD18^−/− (human colon cancer) cells were from Dr. Tadahiro Shiomi [27 (link)] and as used earlier [29 (link)]. Cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 1x Penicillin/Streptomycin sulfate (Gibco) [57 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and were used prior to ten passages. CPT (Sigma) was used at concentrations and time periods indicated. Antibodies against the following targets were used: FANCD2, Rad51, 53BP1, GAPDH (all from Santa Cruz Biotechnology) BRCA2, RAD18 (Bethyl Laboratories) and γH2AX (Millipore). To express wild-type or the RING mutant RAD18, HCT116−/− cells were transfected with pcDNAmycRAD18 and pcDNARAD18 C28F, respectively.

Tripathi K., Mani C., Clark D.W, & Palle K. (2016). Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery. Oncotarget, 7(11), 12537-12553.

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Chromatin Immunoprecipitation Assay Protocol

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CCNC (A301-989A, Bethyl Laboratories), CDK8 (4106S, Cell Signaling Technology), MED12 (4529S, Cell Signaling Technology), MED13 (A301-278A, Bethyl Laboratories), MED24 (A301-472A, Bethyl Laboratories), MED25 (sc-393759, Santa Cruz Biotechnology), BRCA2 (A303-434A, Bethyl Laboratories), Vinculin (#V9131, Sigma), HA (#H3663, Sigma), β-tubulin (#T5168, Sigma), GFP (sc-9996, Santa Cruz Biotechnology), RAD51 (ab63801, Abcam), γH2AX (05-636l, Millipore Sigma).

Tang M., Pei G., Su D., Wang C., Feng X., Srivastava M., Chen Z., Zhao Z, & Chen J. (2021). Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2. Nucleic Acids Research, 49(13), 7476-7491.

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Whole-cell and Nuclear Protein Extraction

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Whole-cell extracts were prepared by lysing cells in NETN450 lysis buffer (450 mM NaCl, 20 mM Tris-HCl, pH 7.8, 0.5% NP-40, 1 mM EDTA, and dH₂O). For nuclear extracts, cells were lysed in Protein Extraction (PEB; 0.5% Triton X, 20 mM Hepes, pH 7, 100 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, and dH₂O) on ice for 20 min followed by spinning at 5,000 rpm for 10 min to remove the cytoplasmic extract. Cell pellets were washed once with PBS followed by lysing in NETN 400 lysis buffer (400 mM NaCl, 20 mM Tris-HCl, pH 7.8, 0.5% NP-40, 1 mM EDTA, and dH₂O) for 1 h at 4°C to generate the nuclear extract. All lysis buffers were supplemented with protease inhibitor and phosphatase inhibitor. Antibodies used for Western blot were RFWD3 (Bethyl; A301-397A; 1:2,500), BRCA2 (Bethyl; A300-005A; 1:3,000), SD118 (Calbiochem; OP107; 1:2,500), GAPDH (Santa Cruz; SC-25778; 1:4,000), GAPDH (BioLegend; 919501; 1:4,000), RAD51 (Santa Cruz; SC-8349; 1:2,500), HA (BioLegend; 901514; 1:3,000), LaminB1 (Cell Signaling; 12596; 1:3,000), pRPA32 S4/S8 (Bethyl; A300-245A; 1:2,500), pRPA S33 (Sigma; PLA0210; 1:2,500), RPA34 (Calbiochem; NA18; 1:3,000), α-Tubulin (Santa Cruz; SC-5286; 1:3,000), Mre11 (Genetex; GTX70212; 1:3,000), and SMARCAL1 (Bethyl; A301-616A; 1:2,500).

Duan H., Mansour S., Reed R., Gillis M.K., Parent B., Liu B., Sztupinszki Z., Birkbak N., Szallasi Z., Elia A.E., Garber J.E, & Pathania S. (2020). E3 ligase RFWD3 is a novel modulator of stalled fork stability in BRCA2-deficient cells. The Journal of Cell Biology, 219(6), e201908192.

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