ID8-luc cells grown on glass coverslips were pretreated for 6 h with the known inducer of double-strand DNA breaks, cisplatin (0.5 µM; Sigma-Aldrich). The cells were then washed with PBS, and treated with vehicle, panobinostat, olaparib or panobinostat/olaparib for 24 h as described above. Following cessation of drug treatment, the cells were fixed, stained with mouse monoclonal anti-BRCA1 (1:100 MilliporeSigma, Burlington, MA) or rabbit polyclonal anti-RAD51 (1:100 MilliporeSigma), with acquisition and analysis of fluorescent images as previously described [39] (link).
Anti rad51
Manufactured by Merck Group
Sourced in United Kingdom, Morocco
Anti-RAD51 is a laboratory reagent used in scientific research. It is a monoclonal antibody that specifically binds to the RAD51 protein, which is involved in DNA repair processes. The core function of Anti-RAD51 is to detect and quantify the presence of RAD51 in biological samples, enabling researchers to study the role of RAD51 in various cellular processes.
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Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti brdu, by BD (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti γh2ax, by Merck Group (2 mentions)
Cisplatin, by Merck Group (1 mentions)
Anti 53bp1, by Novus Biologicals (1 mentions)
Dmi8 inverted fluorescent microscope, by Leica (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Anti ku70 80, by Abcam (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Ecl plus film, by GE Healthcare (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Anti lc3, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Anti ph2ax, by Cell Signaling Technology (1 mentions)
Anti rad52, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti myc, by Merck Group (1 mentions)
20 protocols using anti rad51
1
BRCA1 and RAD51 Localization in DNA Damage
2
Immunofluorescence Imaging of DNA Damage Markers
3
Protein Expression Analysis by Western Blotting
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Total cell lysates were separated in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane, blocked with 5% nonfat milk for one hour at room temperature, and probed with a primary antibody overnight at 4°C. Primary antibodies used included anti‐LC3 (1:5000 dilution, Sigma), anti‐Rad51, anti‐Ku70/80, anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1:1000, 1:500, and 1:1000 dilutions, respectively; Abcam, Cambridge, UK), and γ‐H2AX (1:1000 dilution, Cell Signaling Technologies, Danvers, MA, USA). The membranes were incubated with horseradish peroxidase‐conjugated secondary antibody at a dilution of 1:2000 for one hour at room temperature. Protein bands were visualized using ECL Western Blotting Detection Reagents (Millipore, Billerica, MA, USA) and exposed to an ECL Plus film (GE Healthcare, Piscataway, NJ, USA).
4
Protein Extraction and Western Blot
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Preparation of samples: total cell protein extracts were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific). Anti-ß-actin antibody (AC-15) was purchased from Sigma, anti-rad51 (#8875), anti-rad52 (#3425), anti-p21 (#2947) and anti-p-H2AX (#9718) from Cell Signaling Technology, and anti-p53 (DO-1) from Thermo Fisher Scientific and anti-MVP (#ALX-801–005) from Enzo Life Science. Secondary anti-mouse (#7076) and anti-rabbit (#7074) horseradish peroxidase-labeled antibodies were obtained from Cell Signaling Technologies.
5
Immunoblotting of DNA Repair Proteins
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For the immunoblots of site-specific crosslinking experiments: anti-Rad51 (Rb 1:10,000; provided by Hiroshi Iwasaki); anti-Sfr1 (Rb 1:5,000; provided by Hiroshi Iwasaki). For the immunoblots of cellular proteins: anti-MYC (Rb 1:1,000; Sigma Aldrich C3956), anti-Rad51 (Rat 1:10,000; provided by Hiroshi Iwasaki), and anti-tubulin (Mu 1:10,000; Sigma Aldrich T5168). For the detection of Sfr1 in the Affi-gel assay: anti-Sfr1 (Rb 1:5,000; provided by Hiroshi Iwasaki). For IP of Rad51 complexes, anti-Rad51 (Rb; provided by Hiroshi Iwasaki) was used, and for detection by immunoblotting: anti-Rad51 (Rat 1:10,000; provided by Hiroshi Iwasaki), anti-Sfr1 (Mu 1–5 and 76–80, 1:1000 each; provided by Hiroshi Iwasaki), anti-Rad55 (1:5,000; provided by Hiroshi Iwasaki), and anti-Rad57 (1:5,000; provided by Hiroshi Iwasaki). For IP of Rad57 complexes, anti-Rad57 (Rb; provided by Hiroshi Iwasaki) antibody was used.
6
In vitro Co-immunoprecipitation Assay
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In vitro co-immunoprecipitation assays were performed as follows. Each protein (0.5 µg) as indicated was mixed in 10 µl buffer (25 mM Tris-OAc [pH 7.5], 100 mM KCl, 0.5% NP40, 3 mM Mg-acetate) for 10 min at room temperature followed 20 min at 4°C. To these samples were added M2 FLAG tag antibodies in 90 µl buffer, and the mixtures were incubated for 1 h at 4°C. Dynabeads Protein G (Invitrogen) were then added, and the samples were incubated for 1 h incubation at 4°C. Immunocomplexes were washed three times with 200 µl buffer, and bead-bound proteins were eluted with SDS loading buffer and subjected to 10% SDS-PAGE. Protein complexes were detected by western blotting using anti-Rad51, anti-Sfr1, or anti-M2 antibody (Sigma).
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In vitro co-immunoprecipitation assays were performed as follows. Each protein (0.5 µg) as indicated was mixed in 10 µl buffer (25 mM Tris-OAc [pH 7.5], 100 mM KCl, 0.5% NP40, 3 mM Mg-acetate) for 10 min at room temperature followed 20 min at 4°C. To these samples were added M2 FLAG tag antibodies in 90 µl buffer, and the mixtures were incubated for 1 h at 4°C. Dynabeads Protein G (Invitrogen) were then added, and the samples were incubated for 1 h incubation at 4°C. Immunocomplexes were washed three times with 200 µl buffer, and bead-bound proteins were eluted with SDS loading buffer and subjected to 10% SDS-PAGE. Protein complexes were detected by western blotting using anti-Rad51, anti-Sfr1, or anti-M2 antibody (Sigma).
7
Comprehensive Antibody Staining Protocol
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Anti JMJD6 (Santa Cruz, sc28349), anti UBF (Bethyl, A301-859A), anti histone H3 (Abcam, Ab1791), anti BrdU (Sigma, B2531), anti gamma H2AX (Cell signaling, 9718(20E3)), anti GAPDH (Chemicon, MAb374), anti myc (Santa Cruz, sc-40), anti Treacle (Santa Cruz, sc374536), anti V5 (Cell Signaling, 12032), anti NBS1 (Sigma, PLA0179), anti phospho ATM (S1981) (Cell signaling, 10H11.E12).Anti Rad51 (Millipore, PC130), Anti RPA S33 (Bethyl, A306-246A-T)
8
Immunofluorescence Assay of Ovarian Cancer Cells
9
PARP Inhibitor and HDAC Inhibitor Effects
10
Homologous Recombination Assay in Ovarian Cancer
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