Anti rad51antibody

Manufactured by Cell Signaling Technology

The Anti-Rad51 Antibody is a research-use only product that targets the Rad51 protein. Rad51 is a key protein involved in the process of homologous recombination, which is a critical DNA repair mechanism in eukaryotic cells. This antibody can be used to detect and study the Rad51 protein in various experimental systems.

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Lab products found in correlation

Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions) Poly d lysine coated chamber slides, by Merck Group (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions) Lsm 880 confocal microscope, by Zeiss (1 mentions) Prolong gold antifade reagent, by Vector Laboratories (1 mentions)

3 protocols using anti rad51antibody

Quantifying DNA Damage Response to AZD1775 and Olaparib

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Cells were treated with AZD1775 and/or olaparib for 48 hours in poly-D-lysine coated chamber slides (Sigma-Aldrich, St Louis, MO). Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% Triton X in PBS for 10 minutes. Cells were incubated in blocking solution (5% milk in 0.05% Triton X-PBS) for 30 minutes followed by an overnight incubation in anti-Rad51antibody (Cell Signaling Technology) at a dilution of 1:500. After several washes, Alexa Fluor 488 conjugated anti-rabbit antibody (1:500) was applied for 1 hour. ProLong Gold Antifade Mountant with DAPI (Life Technologies, Waltham, MA) was used for mounting. Images were acquired using an inverted epifluorescence microscope at 100X magnification.

Garcia T.B., Snedeker J.C., Baturin D., Gardner L., Fosmire S.P., Zhou C., Jordan C.T., Venkataraman S., Vibhakar R, & Porter C.C. (2017). A small molecule inhibitor of WEE1, AZD1775, synergizes with olaparib by impairing homologous recombination and enhancing DNA damage and apoptosis in acute leukemia. Molecular cancer therapeutics, 16(10), 2058-2068.

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Immunoaffinity Purification of Ubiquitinated RAD51

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For immunoaffinity purification of endogenous ubiquitinated RAD51, 10 cm tissue culture dish was washed with cold PBS and lysed using lysis buffer A (50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 1% Triton X100) with fresh 1 mM NEM, protease and phosphatase inhibitors (Roche). 1 mg of protein was incubated overnight at 4 °C with anti-RAD51 antibody (Cell Signaling). Protein A sepharose beads (Pierce™)were added to these lysates and rotated at 40C for 2 h, washed 3 times with lysis buffer A and resuspended in 2× Laemmli buffer (Bio-Rad). The immunoprecipitates were divided into 2 parts; 90% of immunoprecipitates were probed for ubiquitin antibody whereas 10% of immunoprecipitates were probed with RAD51 antibody.

Liu Y., Awadia S., Delaney A., Sitto M., Engelke C., Patel H., Calcaterra A., Zelenka-Wang S., Lee H., Contessa J., Neamati N., Ljungman M., Lawrence T.S., Morgan M.A, & Rehemtulla A. (2020). UAE1 inhibition mediates the unfolded protein response, DNA damage and caspase-dependent cell death in pancreatic cancer. Translational Oncology, 13(11), 100834.

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Immunofluorescence Imaging of Rad51 and RPA

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Cell samples were prepared as previously described (46 (link)). Briefly, U2OS cells plated on LabTek chamber slides (Thermo Fisher Scientific) were incubated in CSK buffer for 10 min. The cells were fixed with 4% paraformaldehyde for 20 min. Cells were incubated with the anti-Rad51 antibody (cat no. 8875; Cell Signaling Technology) or anti-RPA antibody (cat no. ab2175; Abcam) at 4°C overnight. After 30 min incubation with Alexa Fluor-conjugated secondary antibody, cells were mounted with ProLong Gold antifade reagent (Vector Laboratories). Confocal images were obtained using an LSM880 confocal microscope (Carl Zeiss). The images were analyzed using ZEN2.1 software.

Oh J.M., Kang Y., Park J., Sung Y., Kim D., Seo Y., Lee E.A., Ra J.S., Amarsanaa E., Park Y.U., Lee S.Y., Hwang J.M., Kim H., Schärer O., Cho S.W., Lee C., Takata K.I., Lee J.Y, & Myung K. (2023). MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1. Nucleic Acids Research, 51(11), 5584-5602.

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