Rabbit anti rad51

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Rabbit anti-RAD51 is an antibody product that detects the RAD51 protein. RAD51 is a key protein involved in DNA repair processes. This antibody can be used to study the expression and localization of RAD51 in various experimental systems.

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Lab products found in correlation

Mouse anti brca1, by Santa Cruz Biotechnology (2 mentions) Mouse anti rpa34 20, by Merck Group (2 mentions) Rabbit anti flag, by Merck Group (2 mentions) Rabbit anti 53bp1, by Novus Biologicals (2 mentions) Rabbit anti γh2ax, by Cell Signaling Technology (2 mentions) Rabbit anti gfp, by Abcam (1 mentions) Mouse anti nsp1 antibody, by Abcam (1 mentions) Horseradish peroxide conjugated anti rabbit antibody, by Promega (1 mentions) Goat anti atr, by Santa Cruz Biotechnology (1 mentions) Rabbit anti atm, by Cell Signaling Technology (1 mentions) Rabbit anti parp 1, by Abcam (1 mentions) Mouse anti chk1, by Santa Cruz Biotechnology (1 mentions) Mouse anti cyclin e, by Santa Cruz Biotechnology (1 mentions) Rabbit anti arid1a, by Cell Signaling Technology (1 mentions) Mouse anti ku70, by Abcam (1 mentions) Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 chicken anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Fluorescence microscope, by Thermo Fisher Scientific (1 mentions) Mouse anti γh2ax 05 636, by Merck Group (1 mentions) Fitc conjugated donkey anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)

Spelling variants of this product

RAD51 (109 citations) Anti-RAD51 (55 citations) Rabbit anti-RAD51 H-92 (4 citations) Rabbit anti-RAD51 polyclonal antibody (3 citations) Rabbit anti-TM (2 citations) Rabbit anti-Rad51 (H-92) antibody (2 citations) Rabbit anti-Rad51 antibody (2 citations)

22 protocols using rabbit anti rad51

Visualizing DNA Damage Markers in U2OS Cells

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For staining lacO tags, lentivirally infected U2OS-lacO/GFPLacI cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature (RT), permeabilized in 0.5% NP40/PBS buffer for 10 min at RT, blocked in 1% bovine serum albumin (BSA) in PBS, and incubated with rabbit anti-GFP (1:500) (Abcam, 290). For staining RAD51 foci, lentivirally infected U2OS cells were permeabilized in Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH at pH 7.9, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 5 min at RT and fixed in 3% paraformaldehyde (in PBS, 2% sucrose) for 10 min at RT, followed by permeabilization in Triton X-100 buffer for 10 min RT. Cells were blocked in 1% BSA/PBS, followed by incubation with rabbit anti-RAD51 (4 μg/ml) (Santa Cruz Biotechnology, 8349). Primary antibodies were detected with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibodies (1:100) (Jackson Laboratories). DNA was stained with DAPI (0.2 μg/ml) to score micronuclei.

Ramamoorthy M, & Smith S. (2015). Loss of ATRX suppresses resolution of telomere cohesion to control recombination in ALT cancer cells. Cancer cell, 28(3), 357-369.

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Rad51 Protein Analysis in Yeast Strains

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Western blottin was performed to check Rad51 levels in NRY1, NRY2, and TSY17 strains. Protein samples were loaded on an SDS polyacrylamide gel. A polyvinylidene difluoride (PVDF) membrane was used for the transfer as described earlier (39 (link)). The primary antibodies used were mouse anti-Act1 (Abcam), rabbit anti-Rad51 (Santa Cruz), and mouse anti-Hsp82 (Calbiochem) at 1:5,000 dilutions. For subcellular fractionation, we used anti-Pgk1 antibody (Novus Biologicals) and mouse anti-Nsp1 antibody (Abcam) at 1:3,000 and 1:5,000 dilutions, respectively. For secondary antibodies, horseradish peroxide-conjugated anti-rabbit antibody (Promega) and anti-mouse antibody (Santa Cruz Biotechnology Inc., CA, USA) were used at 1:10,000 dilutions. The Western blots were developed using a chemiluminescent detection system (Pierce). Every experiment was repeated at least 3 times, and band intensities were quantified by using Image J software. Mean relative densities were plotted using GraphPad prism.

Suhane T., Bindumadhavan V., Fangaria N., Nair A.S., Tabassum W., Muley P., Bhattacharyya M.K, & Bhattacharyya S. (2019). Glu-108 in Saccharomyces cerevisiae Rad51 Is Critical for DNA Damage-Induced Nuclear Function. mSphere, 4(2), e00082-19.

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Immunoblotting Analysis of DNA Damage Response Proteins

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Cells were left to reach 70–80% confluence before cell lysates were harvested. Primary antibodies: rabbit anti-ATM (1:500, #2873, Cell Signalling), goat anti-ATR (1:500, # 1887, Santa Cruz), mouse anti-CHK1 (1:500, # 8408, Santa Cruz), mouse anti-Cyclin E (1:500, # 247, Santa Cruz), rabbit anti-DNA-PKcs (1:500, # 9051, Santa Cruz), mouse anti-Ku70 (1:500, #3114, Abcam), rabbit anti-Ku80 (1:500, #80592, Abcam), rabbit anti-PARP-1 (1:500, # 227244, Abcam), rabbit anti-RAD51 (1:500, # 8349, Santa Cruz), rabbit anti-XRCC1 (1:500, #11429, Santa Cruz), rabbit anti-ARID1A (1:1000, #12354, Cell Signalling). Ponceau-S staining was used to ensure equal loading of the protein.

Bradbury A., O’Donnell R., Drew Y., Curtin N.J, & Sharma Saha S. (2020). Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting. Cancers, 12(7), 1939.

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Evaluating miR-7-5p's Impact on DNA Repair

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H446R cells were transfected with miR-7-5p mimic or control miRNA for 24 h, and then cells were treated with 25 μg/ml doxorubicin for another 24 h. The cells were fixed and stained with mouse anti-BRCA1 (1:100 dilution; Santa Cruz, USA) and rabbit anti-Rad51 (1:66.67 dilution; Santa Cruz, USA). Goat anti-mouse IgG-Alexa Fluor® 594 or chicken anti-rabbit IgG-Alexa Fluor® 488 (1:400 dilution; Invitrogen, USA) was used as the secondary antibody. Photos were taken with a fluorescence microscope (Life Technologies, USA) at a magnification of 200 × .

Lai J., Yang H., Zhu Y., Ruan M., Huang Y, & Zhang Q. (2019). MiR-7-5p-mediated downregulation of PARP1 impacts DNA homologous recombination repair and resistance to doxorubicin in small cell lung cancer. BMC Cancer, 19, 602.

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Immunofluorescence Analysis of DNA Damage Markers

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Cells were fixed in 2% paraformaldehyde in PBS for 10 min at RT, permeabilized in 0.5% NP-40/PBS for 10 min at RT, blocked in 1% BSA/PBS, and incubated with mouse anti-γH2AX #05-636 (0.2 µg/ml) (Millipore) and rabbit anti-53BP1 NB 100–304 (0.1 µg/ml) (Bovus Biologicals) or mouse anti-PML sc966 (2.0 µg/ml) (Santa Cruz) and rabbit anti-TRF1 415 serum (1:1000). For staining RAD51 foci, cells were permeabilized in Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH at pH 7.9, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 5 min at RT, fixed in 3% paraformaldehyde (in PBS, 2% sucrose) for 10 min at RT, permeabilized in Triton X-100 buffer for 10 min RT, blocked in 1% BSA/PBS, and incubated with rabbit anti-RAD51 (4 µg/ml) (Santa Cruz Biotechnology, 8349) for 2hr. Primary antibodies were detected with FITC-conjugated donkey anti-rabbit antibodies (1:100) (Jackson Laboratories). DNA was stained with DAPI (0.2 µg/ml).

Daniloski Z, & Smith S. (2017). Loss of tumor suppressor STAG2 promotes telomere recombination and extends the replicative lifespan of normal human cells. Cancer research, 77(20), 5530-5542.

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Western Blot Analysis of BRCA1 and Rad51

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Proteins from whole cell lysates were separated by 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was then blocked and incubated with rabbit anti-BRCA1 (1:600 dilution; Proteintech Group, Chicago, USA), rabbit anti-Rad51 (1:750 dilution; Santa Cruz, Texas, USA) or mouse anti β-actin (1:4000 dilution; Proteintech Group) at 4 °C overnight. Peroxidase-labeled anti-rabbit or anti-mouse secondary antibodies (1:5000 dilution; KPL, Gaithersburg, MD, USA) were used to detect the specific protein-antibody complex. Bands were visualized by an ECL substrate (Bio-Rad) and scanned using Image Lab Software in Molecular Imager® ChemiDoc^TM XRS+ (Bio-Rad). Equal protein loading was verified by β-actin signal.

Xiao M., Cai J., Cai L., Jia J., Xie L., Zhu Y., Huang B., Jin D, & Wang Z. (2017). Let-7e sensitizes epithelial ovarian cancer to cisplatin through repressing DNA double strand break repair. Journal of Ovarian Research, 10, 24.

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Immunoprecipitation Profiling of DNA Repair Proteins

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Immunoprecipitation experiments were performed using 3×106 293T cells. IP buffer (0.5% Triton X-100, 50mM Tris HCl pH 8.0, 150 mM NaCl, EGTA 1 mM) supplemented with phosphatase, protease inhibitors and benzonase was used for cells lysis. Two mL of lysate were incubated overnight at 4°C with 20 μl of Anti-Flag M2 magnetic beads (Sigma) or Anti-RPA32 conjugated Dynabeads (2μg of MABE285 anti-RPA34–20 mouse (Millipore) with 40μl of Dynabeads protein G (Invitrogen). After extensive washing in IP buffer, proteins were released in 2X Laemmli buffer buffer and subjected to Western blotting.
Blots were incubated with primary antibodies: rabbit anti-FLAG (Sigma-Aldrich); rabbit anti-RPA70 (Genetex); mouse anti-RPA34–20 (Millipore); rabbit anti-pS440/467WRN (Abgent; custom-made); rabbit anti-Lamin B1 (Abcam); rabbit anti-RAD51 (Santa Cruz); rabbit anti-GST (Calbiochem). Blots were detected using the Western blotting detection kit WesternBright ECL (Advansta) according to the manufacturer’s instructions. Quantification was performed on scanned images of blots using Image Lab software, and values shown on the graphs represent normalization of the protein content evaluated through Lamin B1 or Immunoprecipitated protein immunoblotting.

Noto A., Valenzisi P., Fratini F., Kulikowicz T., Sommers J.A., Di Feo F., Palermo V., Semproni M., Crescenzi M., Brosh RM J.r., Franchitto A, & Pichierri P. (2023). PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES. bioRxiv.

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Optimized Protein Detection and Characterization

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After preparing cell lysates, the protein concentration was determined with a bicinchoninic acid (BCA) kit. An equal amount of total protein content (20 μg) was resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) via a trans-blot semi-dry transfer cell system or wet-transfer using a mini-trans-blot electrophoretic transfer cell system (Bio-Rad Laboratories). For immunoblots, the primary antibodies used were rabbit anti-phospho-histone H2A. X (Cell Signaling Technology, Inc., catalog no. 9718S), rabbit ani-p21 Waf1/Cip1 (12D1) (Cell Signaling Technology, Inc., catalog no. 2947), mouse anti-p53 (Santa Cruz Biotechnology, catalog no. DO-1), mouse anti-phospho-ATM (Santa Cruz Biotechnology, catalog no. sc-47739), rabbit anti-53BP1 (Novus Biologicals, catalog no. NB100-904), mouse anti-BRCA1 (Santa Cruz Biotechnology, catalog no. sc-6954), and rabbit anti-RAD51 (Santa Cruz Biotechnology, catalog no. sc-8349). The secondary antibodies used were anti-rabbit IgG conjugated to horseradish peroxidase (HRP) and anti-mouse IgG conjugated to HRP (Santa Cruz Biotechnology). All blots were stripped and re-probed with monoclonal anti-α-tubulin antibody (DM1A, Sigma-Aldrich) as a loading control. Signals were visualized by ECL plus Western Blotting Detection Reagents (GE Healthcare) and a Kodak x-omat 1000A Film Processor.

Giannakakis A., Tsifintaris M., Gouzouasis V., Ow G.S., Aau M.Y., Papp C., Ivshina A.V, & Kuznetsov V.A. (2024). KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer. Frontiers in Oncology, 14, 1227151.

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Immunofluorescence Staining of DNA Damage Markers

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Healthy donor PBMCs designated for IF were treated in parallel with those designated for flow cytometry throughout stimulation and drug exposure. Cells were cytospun onto silylated slides (CEL Associates; Pearland, TX, USA) and were stained with mouse anti-human γH2AX (1:200, Abcam) and rabbit anti-MRE11 (1:200, Abcam), and rabbit anti-RAD51 (1:50, Santa Cruz Biotechnology; Santa Cruz, CA, USA) using our standard laboratory immunofluorescence protocol [21 (link)]. Alexa Fluor 488 goat-anti mouse IgG and Alexa Fluor 488 goat-anti rabbit IgG (1:500, Invitrogen) were used for IF detection. The slides were mounted with Vectashield mounting medium with DAPI (Vector Labs; Burlingame, CA, USA) and images were collected as Z stacks using a Zeiss LSM 780 confocal microscope at 1000× magnification. Images were analyzed using Zeiss LSM Image Browser version 4.2.0.121 software.

Lee J.M., Gordon N., Trepel J.B., Lee M.J., Yu M, & Kohn E.C. (2015). Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer. Journal of Translational Medicine, 13, 239.

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Immunofluorescent Analysis of RAD51 Expression

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Cells treated with the specified drugs were cultured on coverslips. After 48 hours, they were fixed with 4% paraformaldehyde and blocked with a 5% bovine serum albumin-phosphate-buffered solution. They were then stained with rabbit anti-RAD51 (Santa Cruz Biotechnology). After washing with phosphate-buffered saline, they were treated with secondary antibodies. Slides were mounted in Vectashield with DAPI. Analyses were performed using an immunofluorescence microscope (Leica).

Wang Z., Dai Z., Wang B., Gao Y., Gao X., Wang L., Zhou S., Yang L., Qiu X, & Liu Z. (2021). Targeting c-MET to Enhance the Efficacy of Olaparib in Prostate Cancer. OncoTargets and therapy, 14, 4383-4389.

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