Anti brca1 d9

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-BRCA1 (D9) is a monoclonal antibody that recognizes the BRCA1 protein. BRCA1 is a tumor suppressor protein involved in DNA repair and cell cycle regulation. The antibody can be used for the detection and analysis of BRCA1 in various applications.

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Lab products found in correlation

Anti β actin, by Merck Group (2 mentions) Anti flag m2, by Merck Group (2 mentions) Anti β actin a1978, by Merck Group (1 mentions) Anti ha h9658, by Merck Group (1 mentions) Anti ub p4d1, by Santa Cruz Biotechnology (1 mentions) Anti flag f1804, by Merck Group (1 mentions) Anti 53bp1 a300 272a, by Fortis Life Sciences (1 mentions) Ha flag usp13, by Addgene (1 mentions) Pgex 4t 2 vector, by Takara Bio (1 mentions) Anti γh2ax 05 636, by Merck Group (1 mentions) Anti mdc1, by Merck Group (1 mentions) Chemiluminescence kit, by Merck Group (1 mentions) A2228, by Merck Group (1 mentions) Sc 6954, by Santa Cruz Biotechnology (1 mentions) Anti parp, by Cell Signaling Technology (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Horseradish peroxidase linked secondary antibody, by Cytiva (1 mentions) Peroxidase affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions) Anti human igg antibody, by Abcam (1 mentions) Alexa fluor 488 affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)

7 protocols using anti brca1 d9

Characterization of USP13 Regulation

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HA-FLAG-USP13 was purchased from Addgene (Plasmid #22568, provided by Dr Wade Harper) and subcloned into pGEX-4 T-2 vector (Clontech). USP13 site mutants were generated by site-directed mutagenesis (Stratagene).
The anti-USP13 (GTX118595, dilution: 1:500) and anti-Rad51 (N1C2, dilution: 1:200) antibodies were purchased from Genetex. Anti-Ub (P4D1, dilution: 1:500), anti-RPA32 (9H8, dilution: 1:200) and anti-BRCA1 (D9, dilution: 1:200) antibodies were purchased from Santa Cruz Biotechnology. Anti-γH2AX (05-636, dilution: 1:500), anti-FK2 (04-263, dilution: 1:500) and anti-MDC1 (05-1572, dilution: 1:200) were purchased from Millipore. Anti-RAP80 (A303-763A, dilution: 1:500) and anti-53BP1 (A300-272A, dilution: 1:500) were purchased from Bethyl Laboratories. Anti-FLAG (F1804, dilution: 1:1,000), anti-HA (H9658, dilution: 1:1,000), and anti-β-actin (A1978, dilution: 1:2,000) antibodies were purchased from Sigma. Anti-RNF8 (ab4183, dilution: 1:500) was purchased from Abcam.

Li Y., Luo K., Yin Y., Wu C., Deng M., Li L., Chen Y., Nowsheen S., Lou Z, & Yuan J. (2017). USP13 regulates the RAP80-BRCA1 complex dependent DNA damage response. Nature Communications, 8, 15752.

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Western Blot Analysis of BRCA1 and PARP

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Western blot analysis was performed as described previously [23 (link)]. Briefly, cell extracts were prepared using Laemmli buffer. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The following antibodies were used for immunoblotting: anti-β-actin (dilution 1:10000; A2228, Sigma-Aldrich), anti-BRCA1 (dilution 1:300; MAB22101, R&D systems (Minneapolis, MN, USA)), anti-BRCA1 (D-9)(dilution 1:100; sc-6954, Santa Cruz Biotechnology (Dallas, TX, USA)), and anti-PARP (dilution 1:2000; #9542, Cell Signaling Technology (Danvers, MA, USA)). Immune complexes were detected using a horseradish peroxidase-linked secondary antibody (Cytiva, Tokyo, Japan), and immunoreactive proteins were visualized using a chemiluminescence kit (Merck, Branchburg, NJ, USA) and the ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Image quantification was performed using ImageJ software (NIH, Rockville, MD, USA).

Sasaki Y., Inouchi T., Nakatsuka R., Inoue A., Masutani M, & Nozaki T. (2024). Activated NAD+ biosynthesis pathway induces olaparib resistance in BRCA1 knockout pancreatic cancer cells. PLOS ONE, 19(4), e0302130.

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Immunofluorescence and Western Blotting Antibodies

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Antibodies used for immunofluorescence (IF) staining experimentations: Anti-Ki-67 (EMD Millipore, #MAB4190); Anti-C23 (H-250; Santa Cruz, #sc-13057); Anti-γH2AX (11 (link)); Anti-53BP1 (11 (link)); Anti-UBF (F-9) (Santa Cruz, #sc-13125); Anti-BRCA1 (D9; Santa Cruz, #sc-6954); Anti-Rad51 (14B4; Novus Biologicals; NB100-148); Anti-RNF169 (12 (link)); Anti-Flag (M2; Sigma-Aldrich, #F3165); Anti-HA.11 Epitope Tag Antibody (BioLegend, #901514); Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, #115-585-166); Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, #111-545-144).
Antibodies used for western blotting: Anti-DYRK1B (H-6; Santa Cruz, #sc-390417); Anti-RNF169 (12 (link)); Anti-Flag (M2; Sigma-Aldrich, #F3165); Anti-HA.11 Epitope Tag Antibody (BioLegend, #901514); Anti-β-Actin (Sigma-Aldrich, #A5441); Anti-Human IgG antibody (Abcam, #ab2410); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 405 (Thermo Fisher Scientific, # A31556); Peroxidase AffiniPure Rabbit Anti-mouse IgG+IgM (H+L) (Jackson ImmunoResearch, #315-035-048); Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, #111–035-144).

Dong C., An L., Yu C.H, & Huen M.S. (2021). A DYRK1B-dependent pathway suppresses rDNA transcription in response to DNA damage. Nucleic Acids Research, 49(3), 1485-1496.

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Antibody Characterization for DNA Damage Response

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Antibodies used in this study were as follows: anti-USP15 (A300-923A, WB: 1:1000), anti-BARD1 (A300-263A, WB: 1:1000; IF: 1:200), and anti-RAP80 (A300-764A, WB: 1:1000; IF: 1:100) were purchased from Bethyl Laboratory. Anti-HP1γ (MABE656, WB: 1:2000). Mouse anti-γ-H2AX (05-636, IF: 1:500), mouse anti-MDC1 (05-1572, IF: 1:300) and mouse anti-FK2 (04-263, IF: 1:200) were purchased from Millipore. Anti-RPA 70 (#2267, IF: 1:200), rabbit anti-γ-H2AX (#9718, IF: 1:400), and rabbit anti-HA (#3724, WB: 1:2500; IF: 1:300) were from Cell signaling Technology. Anti-Rad51 (GTX100469, IF: 1:200) were from Genetex. Anti-BRCA1 (D9, IF: 1:50) and anti-His tag (sc8036, WB: 1:1000) were from Santa Cruz. Anti-poly PAR (4336-BPC-100, WB: 1:1000) were from Trevigon. Anti-53BP1 (NB100-304, WB: 1:1000; IF: 1:300) were from Novus Biologicals. Anti-RNF8 (14112-1-AP, IF: 1:100) were from Proteintech. Rabbit anti-FLAG (F7425, WB: 1:2500) and mouse anti-FLAG (F3165, WB: 1:2500) were from Sigma-Aldrich. Mouse anti-HA (901501, WB: 1:2500; IF: 1:100) were from BioLegend.

Peng Y., Liao Q., Tan W., Peng C., Hu Z., Chen Y., Li Z., Li J., Zhen B., Zhu W., Li X., Yao Y., Song Q., Liu C., Qi X., He F, & Pei H. (2019). The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors. Nature Communications, 10, 1224.

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Antibody Characterization for DNA Damage Response

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The following antibodies were used in this study: anti-FLAG (F3165, Sigma, St Louis, MO, USA); anti-BRCA1 (D-9, Santa Cruz, Dallas, TX, USA); anti-cyclin A (sc-751 and sc-271645, Santa Cruz); anti-NBS1 (A301-289A, Bethyl, Montgomery, TX, USA); anti-53BP1(612522, BD, San Jose, CA, USA); anti-phospho-ATM (S1981) (4526, Cell Signaling, Danvers, MA, USA); anti-phospho-RPA32 (S4/S8) (A300-245, Bethyl); anti-phospho-53BP1 (S25/29) (2674, Cell Signaling); anti-phospho-53BP1 (S1778) (2675, Cell Signaling); anti-phospho-NBS1 (S343) (3001, Cell Signaling); anti-phospho-SMC1 (S957) (4801, Cell Signaling) anti-phospho-CHK1 (S345) (2348, Cell Signaling); anti-phospho-KAP-1 (S824) (4127, Cell Signaling) and anti-phospho-(Ser/Thr) ATM/ATR substrate antibody (2851, Cell Signaling). Rabbit anti-PTIP antisera were obtained from rabbits immunized with GST-PTIP (residues 590–1 069) fusion protein expressed and purified from Escherichia coli. Antisera were affinity-purified using AminoLink Plus Immobilization and Purification Kit (Pierce, Waltham, MA, USA ). Rabbit polyclonal antibodies against BRCA1, RIF1, ATM, γH2AX, 53BP1 and phospho-CHK2 (T68) were described previously [5 (link), 31–33 ].

Feng L., Li N., Li Y., Wang J., Gao M., Wang W, & Chen J. (2015). Cell cycle-dependent inhibition of 53BP1 signaling by BRCA1. Cell Discovery, 1, 15019-.

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Immunoprecipitation of Brca1 and TPX2

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Cells were lysed in 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.25% NP-40, 10% glycerol, 1 mM DTT, protease inhibitor and phosphatase inhibitor cocktails (Roche, Switzerland). 2 mg lysate were incubated with 1.5 µg anti-Brca1 (D-9, Santa Cruz, USA) or anti-TPX2 (18D5, Santa Cruz) antibodies and immunocomplexes were precipitated using protein-G sepharose beads (GE Healthscare, USA).

Ertych N., Stolz A., Stenzinger A., Weichert W., Kaulfuß S., Burfeind P., Aigner A., Wordeman L, & Bastians H. (2014). Increased microtubule assembly rates mediate chromosomal instability in colorectal cancer cells. Nature cell biology, 16(8), 779-791.

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Detailed Protocols for DNA Damage Response

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The ATM inhibitor KU55933 (a final concentration of 10 μM was used throughout this research) was purchased from Selleck, and the DNA-PKcs inhibitor NU7026 (a final concentration of 10 μM was used), the ATR inhibitor NU6027 (a final concentration of 10 μM was used), and etoposide (a final concentration of 10 μM was used) were purchased from Sigma.
Rabbit polyclonal antibodies anti-BCL10, anti-MALT1, anti-DNA-PKs, anti-BRCA1, anti-MYC, anti-HA, and anti-RAP80 were from Bethyl; mouse monoclonal antibodies anti-γ-H2AX, rabbit polyclonal antibodies anti-RNF168 (ABE367) for IF, anti-RNF168 (06-1130) for IP, and anti-ubiquitin (FK2) were from Millipore; rabbit monoclonal antibody anti-GST (A00865) was from GenScript; mouse monoclonal antibody anti-FLAG (M2) and rabbit polyclonal antibody anti-BCL10 (MK-17 for IF) were from Sigma; mouse monoclonal antibody anti-BRCA1 (D-9) and anti-RNF168 (B-11) for IB were from Santa Cruz. Rabbit polyclonal anti-MDC1 antibody was described before.¹¹ (link) Rabbit polyclonal antibodies against RNF8, RAD51, and UBC13 were gifts from Drs Michael SY Huen, Jun Huang, and Wei Xiao, respectively. The rabbit polyclonal anti-pT91-BCL10 antibody was raised against the phospho-peptide IRREK(pT)QNFLI and affinity-purified (AbMart).
All cell lines were cultured in high-glucose Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C.

Zhao H., Zhu M., Dou G., Zhao H., Zhu B., Li J., Liao J, & Xu X. (2014). BCL10 regulates RNF8/RNF168-mediated ubiquitination in the DNA damage response. Cell Cycle, 13(11), 1777-1787.

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