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Anti brca2

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Anti-BRCA2 is a laboratory tool used to detect and analyze the BRCA2 gene. It is a critical component in genetic research and diagnostics related to BRCA2-associated diseases.

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Lab products found in correlation

Anti brca1, by Santa Cruz Biotechnology (4 mentions) Anti rad51, by Santa Cruz Biotechnology (4 mentions) Anti β actin, by Merck Group (2 mentions) Anti 53bp1, by Novus Biologicals (2 mentions) Anti flag m2, by Merck Group (2 mentions) Anti rnf126, by Santa Cruz Biotechnology (2 mentions) Anti rnf126, by Abcam (2 mentions) Anti rad52, by GeneTex (2 mentions) Anti e2f1, by Santa Cruz Biotechnology (2 mentions) Anti gst, by Santa Cruz Biotechnology (2 mentions) Anti ha, by Fortrea (2 mentions) Fugene hd, by Promega (2 mentions) Anti flag, by Merck Group (2 mentions) Anti rad51, by Abcam (2 mentions) Plko 1 vector, by Addgene (1 mentions) Anti cyclin a antibody, by Santa Cruz Biotechnology (1 mentions) Scramble shrna, by Addgene (1 mentions) Hek293t, by RIKEN Cell Bank (1 mentions) Hepg2, by RIKEN Cell Bank (1 mentions) Hek293t, by Promega (1 mentions)

Close spelling variants of this product

BRCA2 (9 citations)

8 protocols using anti brca2

1

Western Blot Antibody Panel for DNA Repair Proteins

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For WB, the following conditions are used. Anti-RNF126 (Clone C-1; 1:200; Santa Cruz Technology); Anti-RNF126 (Clone 1B5; 1:1000; Abcam); Anti-BRCA1 (Clone D-9; 1:200; Santa Cruz Technology); Anti-RAD51 (Clone H92; 1:200; Santa Cruz Technology); Anti-BRCA2 (clone 5.23; 1:500; EMD Millipore); Anti-RAD52 (Clone 5H9; 1:200; GeneTex); Anti-RPA1 (Clone NA13; 1:100; Calbiochem/EMD Millipore) and anti-RPA2 (Clone NA18; 1:100; Calbiochem/EMD Millipore); Anti-53BP1(Clone 1B9; 1:1000; Novus biologicals); Anti-FLAG M2 (Clone M2; 1:1000; Sigma-Aldrich); Anti-E2F1(Clone KH95; 1:200; Santa Cruz Technology); Anti-HA (Clone 16B12; 1:1000; Covance); Anti-His (Clone H-15; 1:200; Santa Cruz Technology); Anti-GST (Clone B-14; 1:200; Santa Cruz Technology); Anti-Filamin (Clone FLMN01; 1:1000; Pierce); Anti-β-Actin ( Clone AC-74; 1:10000; Sigma-Aldrich). Secondary antibodies were goat-anti-mouse IgG–HRP conjugated and goat-anti-rabbit IgG–HRP conjugated both at 1:5 000 dilutions for immune blotting.
Wang Y., Deng O., Feng Z., Du Z., Xiong X., Lai J., Yang X., Xu M., Wang H., Taylor D., Yan C., Chen C., Difeo A., Ma Z, & Zhang J. (2015). RNF126 promotes homologous recombination via regulation of E2F1-mediated BRCA1 expression. Oncogene, 35(11), 1363-1372.
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2

Western Blot Antibody Panel for DNA Repair Proteins

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For WB, the following conditions are used. Anti-RNF126 (Clone C-1; 1:200; Santa Cruz Technology); Anti-RNF126 (Clone 1B5; 1:1000; Abcam); Anti-BRCA1 (Clone D-9; 1:200; Santa Cruz Technology); Anti-RAD51 (Clone H92; 1:200; Santa Cruz Technology); Anti-BRCA2 (clone 5.23; 1:500; EMD Millipore); Anti-RAD52 (Clone 5H9; 1:200; GeneTex); Anti-RPA1 (Clone NA13; 1:100; Calbiochem/EMD Millipore) and anti-RPA2 (Clone NA18; 1:100; Calbiochem/EMD Millipore); Anti-53BP1(Clone 1B9; 1:1000; Novus biologicals); Anti-FLAG M2 (Clone M2; 1:1000; Sigma-Aldrich); Anti-E2F1(Clone KH95; 1:200; Santa Cruz Technology); Anti-HA (Clone 16B12; 1:1000; Covance); Anti-His (Clone H-15; 1:200; Santa Cruz Technology); Anti-GST (Clone B-14; 1:200; Santa Cruz Technology); Anti-Filamin (Clone FLMN01; 1:1000; Pierce); Anti-β-Actin ( Clone AC-74; 1:10000; Sigma-Aldrich). Secondary antibodies were goat-anti-mouse IgG–HRP conjugated and goat-anti-rabbit IgG–HRP conjugated both at 1:5 000 dilutions for immune blotting.
Wang Y., Deng O., Feng Z., Du Z., Xiong X., Lai J., Yang X., Xu M., Wang H., Taylor D., Yan C., Chen C., Difeo A., Ma Z, & Zhang J. (2015). RNF126 promotes homologous recombination via regulation of E2F1-mediated BRCA1 expression. Oncogene, 35(11), 1363-1372.
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3

BRCA2 C-terminus Functional Analysis

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The HeLa, HepG2, and HEK293T cell lines were obtained from RIKEN Cell Bank and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The following antibodies were used for Western blotting and immunostaining: anti-RAD51 (dilution 1:500–1000 H-92; Santa Cruz Biotechnology, Dallas, TX, USA), anti-BRCA2 (dilution 1:250 OP-95; Merck, Darmstadt, Germany), anti-lamin B1 (dilution 1:1000 PM064; MBL, Nagoya, Japan), anti-FLAG (dilution 1:1000 M2; Merck), anti-α-Tubulin (dilution 1:1000 M175-3; MBL), anti-GFP (dilution 1:1000 598; MBL), and anti-Cyclin A antibody (dilution 1:1000 B8; Santa Cruz Biotechnology).
The FLAG-HA- or FLAG-HA-EGFP-fused BRCA2 C-terminus (3260–3418 aa) and FLAG-HA-fused CTRBD (3260–3331 aa) were stably expressed using the pOZ-N plasmid [28 (link)]. The shRNA-mediated knockdown of BRCA2 was achieved by expressing the target sequence 5′-TACAATGTACACATGTAACAC-3′ in the pLKO.1 vector (donated by David Root; Addgene plasmid no. 10878) and scramble shRNA (donated by David Sabatini; Addgene plasmid no. 1864) [29 (link),30 (link)]. The plasmid DNA was transfected into HEK293T cells using FuGENE HD (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions.
Zhu Z., Kitano T., Morimatsu M., Tanaka A., Morioka R., Lin X., Orino K, & Yoshikawa Y. (2022). BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents. International Journal of Molecular Sciences, 23(7), 4060.
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4

Characterizing BRCA2 and RAD51 Interaction

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Cell culture, antibodies, and generating cell lines HeLa, HepG2, and HEK293T cells (RIKEN, Tokyo, Japan) were grown in Dulbecco's modi ed Eagle's medium supplemented with 10% fetal bovine serum. The following antibodies were used for western blotting: anti-RAD51 (dilution 1:500 H-92; Santa Cruz Biotechnology, Dallas, TX, USA) anti-BRCA2 (dilution 1:250 OP-95; Merck, Darmstadt, Germany), anti-Lamin B1 (dilution 1:1000 PM064; MBL, Nagoya, Japan), anti-FLAG (dilution 1:1000 M2; Merck), anti-α-Tubulin (dilution 1:1000 M175-3; MBL), and anti-GFP (dilution 1:1000 598; MBL). FLAG-HA-or FLAG-HA-EGFP-fused BRCA2 C-terminus (3260-3418 aa) and FLAG-HA-fused C-terminal RAD51-binding domain (3260-3331 aa) were stably expressed using the pOZ-N plasmid 19 . shRNAmediated knockdown of BRCA2 was achieved by expressing the target sequence 5′-TACAATGTACACATGTAACAC-3′ in the pLKO.1 vector, which was donated by David Root (Addgene plasmid no. 10878) and scramble shRNA, which was donated by David Sabatini (Addgene plasmid no. 1864) 20, 21 . Transfections of plasmid DNA into HEK293T cells were conducted using FuGENE HD (Promega, Madison, WI, USA) according to the manufacturer's instructions.
Yoshikawa Y., Morimatsu M., Tanaka A., Morioka R., & Orino K. (2021). BRCA2 C-terminal RAD51-binding Domain Confers Resistance to DNA-damaging Agents.
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5

DNA Damage Foci Quantification

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Cells (4 × 105) were plated, treated with PARPi and ATRi for 12 or 24 h, washed in cold PBS, and cytospun onto a glass slide at 79 × g for 5 min. Cells were treated with 0.5% Triton and fixed in cold methanol, followed by blocking in PBS with 0.1% Triton, 2% bovine serum albumin and 10% milk. Cells were incubated with primary anti-γH2AX antibody (1:500, Millipore), anti-BRCA2 (1:1000, Millipore), anti-Rad51 antibody (1:200, Santa Cruz), and anti-RPA (1:200, Thermo Fisher Scientific) overnight at 4 °C in a humidified chamber after incubation with primary anti-γH2AX antibody (1:500, Millipore) for 2 h at room temperature for double staining. Following washing with TBST (Boston Bioproduct), cells were incubated with secondary antibodies Alexa-488 anti-mouse (1:250; Jackson ImmunoResearch) and Cy3 anti-rabbit (1:250; Jackson ImmunoResearch) for 1 h at room temperature, and slides were mounted with VectaShield (DAPI included, Vector Laboratories). Staining was imaged at ×60 with a Nikon 90i microscope and quantified using the software ImageJ.
Ning J.F., Stanciu M., Humphrey M.R., Gorham J., Wakimoto H., Nishihara R., Lees J., Zou L., Martuza R.L., Wakimoto H, & Rabkin S.D. (2019). Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma. Nature Communications, 10, 2910.
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6

Protein Expression Analysis in Cells

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Cells were lysed in mammalian protein extraction reagent (Pierce). After quantification using a BCA protein assay kit (Pierce), total proteins were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore). Membranes were blocked in 5% non-fat milk (Bio-Rad) and then incubated with anti-CDK7 (Cat No: sc-7344, Santa Cruz Biotechnoloy); anti-BRCA1 (Cat No: sc-6954, Santa Cruz); anti-BRCA2 (Cat No: OP95, Millipore); anti-RAD51 (Cat No: ab213, Abcam); anti-Ku80 (Cat No: MA5–12933, Thermo); anti-Ku70 (Cat No: MA5–15110, Thermo); anti-RNAP II (Cat No: sc-17798, Santa Cruz Biotechnoloy); anti-RNAP II p-Ser5 (Cat No: A300–655A-2, Bethyl); or anti-phospho-H2AX(S139) (Cat No:05–636, Clone No: JBW301, Millipore), followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Anti-b-Tubulin (Cat No: 2128, Clone No: 9F3, CST) or anti-Actin (Cat No: A3854, Sigma) was used for internal loading control. Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling).
Shan W., Yuan J., Hu Z., Jiang J., Wang Y., Loo N., Fan L., Tang Z., Zhang T., Xu M., Pan Y., Lu J., Long M., Tanyi J.L., Montone K.T., Fan Y., Hu X., Zhang Y, & Zhang L. (2020). Systematic Characterization of Recurrent Genomic Alterations in Cyclin-Dependent Kinases Reveals Potential Therapeutic Strategies for Cancer Treatment. Cell reports, 32(2), 107884.
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7

Immunofluorescence Analysis of DNA Damage Response

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Cells were seeded on coverslips and treated with THZ1 and ionizing radiation, or with DMSO, THZ1, olaparib or combination of THZ1 and olaparib, then harvested for immunofluorescent staining. Cells were fixed in solution containing 3% paraformaldehyde and 2% sucrose for 10 min at room temperature, and subsequently permeabilized with 0.5% Triton solution for 5 min at 4°C. Permeabilized cells were incubated with anti-γH2AX antibody (Cat No: ab81299, Abcam), anti-BRCA1 (Cat No: sc-6954, Santa Cruz), anti-BRCA2 (Cat No: OP95, Millipore), or anti-RAD51 (Cat No: ab213, Abcam) in PBST buffer (PBS plus 0.1% Tween-20, 0.02% NaN3) overnight at 4°C. Cells were then washed three times with PBST, then incubated with secondary antibody for 1 hour at room temperature. After four washes with PBST, coverslips were mounted onto glass slides using Vectashield mounting medium containing DAPI (Vector Laboratories) and visualized using an Axiovert 200M inverted microscope (Zeiss).
Shan W., Yuan J., Hu Z., Jiang J., Wang Y., Loo N., Fan L., Tang Z., Zhang T., Xu M., Pan Y., Lu J., Long M., Tanyi J.L., Montone K.T., Fan Y., Hu X., Zhang Y, & Zhang L. (2020). Systematic Characterization of Recurrent Genomic Alterations in Cyclin-Dependent Kinases Reveals Potential Therapeutic Strategies for Cancer Treatment. Cell reports, 32(2), 107884.
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8

Histopathological Evaluation of Tumor Samples

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Excised tumors, kidney and livers were sampled just after sacrifice and representative areas were a) formalin-fixed (24 hours) (Millipore) and paraffin-embedded and (b) snap-frozen in OCT and stored at 80ºC as previously described [32 (link)]. Tissue sections 2μM thick were stained with hematoxilin & eosin and prepared for IHC. Two experienced pathologists (MCGM and EDA) observed the samples under a Leica microscope (Leica Microsystems). IHC was performed as previously described [32 (link)] using the following primary antibodies: anti-pH2AX (Millipore); anti-cleaved PARP (cell signaling); anti-Ki67 (Millipore) and anti-BRCA2 (Millipore). TUNEL assays (Roche) were performed to detect DNA fragmentation and late apoptosis. To quantify the extent of necrosis and the IHC findings, the Dotslide analysis program (Olympus) and the Ariol Image analysis system (Olympus) were used respectively.
Ordóñez J.L., Amaral A.T., Carcaboso A.M., Herrero-Martín D., García-Macías M.D., Sevillano V., Alonso D., Pascual-Pasto G., San-Segundo L., Vila-Ubach M., Rodrigues T., Fraile S., Teodosio C., Mayo-Iscar A., Aracil M., Galmarini C.M., Tirado O.M., Mora J, & de Álava E. (2015). The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin. Oncotarget, 6(22), 18875-18890.
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