Rad51

Manufactured by Santa Cruz Biotechnology

Sourced in United States

RAD51 is a protein involved in DNA repair processes. It plays a crucial role in homologous recombination, a mechanism that repairs double-strand breaks in DNA. RAD51 mediates the search for homologous DNA sequences and promotes the formation of joint molecules, which are essential steps in the homologous recombination pathway.

Automatically generated - may contain errors

Lab products found in correlation

109 protocols using rad51

Antibody-Based Chromatin Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following primary antibodies were used: γ-H2AX (#05–636; EMD Millipore), 53BP1 (ab21083; Abcam), Oct3/4 ((#611203; BD Transduction Laboratories), RPA (NA19L; Calbiochem), Rad51 (sc-8349; Santa Cruz Biotechnology, Inc.), GAPDH (#AB2302; Millipore), Fzr1 (ab3242; Abcam), RNA pol II (provided by Pavel Janscak), Ape1 (NB100–116; Novus), Tdg (provided by P. Schär), hTOPO3α (IE3; a kind gift from Aventis Pharma). For chromatin fractionation, Rad51(sc-8349; Santa Cruz Biotechnology, Inc.), RPA32 (2208S; Cell Signaling), MEK2 (610235; BD Transduction Laboratories), Histone-H3 (ab1791; Abcam). The secondary antibodies used were Alexa Fluor conjugates (Alexa Fluor 488, 594 and 647; Life Technologies) for flow cytometry and immunofluorescence and anti-rabbit and anti-mouse ECL (GE Healthcare) for western blotting.

Ahuja A.K., Jodkowska K., Teloni F., Bizard A.H., Zellweger R., Herrador R., Ortega S., Hickson I.D., Altmeyer M., Mendez J, & Lopes M. (2016). A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells. Nature Communications, 7, 10660.

+ Open protocol

+ Expand

Western Blot Analysis of DNA Damage Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with a buffer containing 67 mM Tris-HCl (pH 6.8) and 2% SDS. Denatured proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes as previously described [50 (link)]. Proteins were stained with Ponceau S (0.1% Ponceau reagent [Sigma, P3504] with 5% acetic acid). Incubation with primary antibodies was conducted overnight at 4°C. Antibodies against the indicated proteins were used as follows: RAD51 (Santa Cruz Biotechnology, Dallas, TX, USA, H-92, sc-8349; 1/200), Cyclin A2 (Santa Cruz Biotechnology, H-432 sc-751; 1/500), P-Chk1 S296 (Cell Signaling, Danvers, MA, USA, #2349; 1/1000), Lamin B (Santa Cruz Biotechnology, M-20 sc-6217; 1/200), GAP120 (Santa Cruz Biotechnology, sc-63; 1/100), CDK4 (Santa Cruz Biotechnology, sc-749; 1/500), Polymerase α (Santa Cruz Biotechnology, sc-5921; 1/200), RAD51 (Santa Cruz Biotechnology, sc-8349; 1/200). Proteins were visualized using EZ-ECL Kit (Biological Industries). Signals were detected using imaging. Quantification was done by densitometry using Image Lab^™ Software (Bio-Rad, Hercules, CA, USA, #1709690). Original uncropped and unadjusted images are included in the S1 Raw images (note that some of the membranes were cropped before incubated with the antibodies in order to be able to evaluate different proteins in a single sample).

Feu S., Unzueta F., Ercilla A., Pérez-Venteo A., Jaumot M, & Agell N. (2022). RAD51 is a druggable target that sustains replication fork progression upon DNA replication stress. PLoS ONE, 17(8), e0266645.

+ Open protocol

+ Expand

Western Blot Analysis of DNA Repair Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with a buffer containing 67 mM Tris-HCl (pH 6.8) and 2% SDS. Denatured proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes as previously described (49) . Proteins were stained with Ponceau S (0.1% Ponceau reagent [Sigma, P3504] with 5% acetic acid). Incubation with primary antibodies was conducted overnight at 4°C. Antibodies against the indicated proteins were used as follows: Rad51 (Santa Cruz Biotechnology, Dallas, TX, USA, H-92, sc-8349; 1/200), Cyclin A2 (Santa Cruz Biotechnology, H-432 sc-751; 1/500), P-Chk1 S296 (Cell Signaling, Danvers, MA, USA, #2349; 1/1000), Lamin B (Santa Cruz Biotechnology, M-20 sc-6217; 1/200), GAP120 (Santa Cruz Biotechnology, sc-63; 1/100), CDK4 (Santa Cruz Biotechnology, sc-749; 1/500), Polymerase α (Santa Cruz Biotechnology, sc-5921; 1/200), Rad51 (Santa Cruz Biotechnology, sc-8349; 1/200). Proteins were visualized using EZ-ECL Kit (Biological Industries). Quantification was done by densitometry using Image Lab TM Software (Bio-Rad, Hercules, CA, USA, #1709690).

Feu S., Unzueta F., Ercilla A., Jaumot M., & Agell N. (2022). Rad51 is a druggable target that sustains replication fork progression upon DNA replication stress.

+ Open protocol

+ Expand

Immunostaining of DNA Damage Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

U2-OS cells were transfected twice with different siRNA and then re-plated on coverslips. Cells were then used for immunostaining 72 hours later. Briefly, cells were first fixed with 3% paraformaldehyde containing 2% sucrose for 10 min, then were permeablized with Triton X-100 solution on ice for 5 min, and stained with different primary antibodies (as indicated later) and the appropriate Alexa-488 (Invitrogen) and Alexa-546 (Invitrogen) conjugated secondary antibodies. Images were taken with an Olympus upright fluorescent microscope. Antibodies used for immunofluorescent staining include: PML (Bethyl, A310-167A); Chk1-pS345 (Cell Signaling, 2348); RPA32-pS4pS8 (Bethyl, A300-245A); BLM (Bethyl, A300-110A); BRCA1 (Bethyl, A301-377A); Rad51 (Santa Cruz, sc-8349); TRF1 (abcam, ab10579); TRF2 (Millipore, 05-521 and Novus, NB110-57130); FANCD2 (Novus, 100-182); S9.6 (Millipore, MABE1095).

Pan X., Chen Y., Biju B., Ahmed N., Kong J., Goldenberg M., Huang J., Mohan N., Klosek S., Parsa K., Guh C.Y., Lu R., Pickett H.A., Chu H.P, & Zhang D. (2019). FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops. Scientific Reports, 9, 19110.

+ Open protocol

+ Expand

Investigating DNA Damage Response in Breast Cancer Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

MDA-MB-468 cells were exposed to either drug-free medium or olaparib and/or selinexor. After different durations of exposure, cells were lysed with RIPA buffer (Millipore Upstate, USA) supplemented with protease inhibitor cocktail. For analysis of phosphorylated protein, a phosphatase inhibitor cocktail (PhosSTOP, Roche, Switzerland) was added to the lysis buffer. 40 μg proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with primary antibodies p-H2AX (Cell Signaling Technology, USA) and RAD51 (Santa Cruz Biotechnology, USA); β-actin was used as a loading control.

Marijon H., Gery S., Chang H., Landesman Y., Shacham S., Lee D.H., de Gramont A, & Koeffler H.P. (2021). Selinexor, a selective inhibitor of nuclear export, enhances the anti-tumor activity of olaparib in triple negative breast cancer regardless of BRCA1 mutation status. Oncotarget, 12(18), 1749-1762.

+ Open protocol

+ Expand

Quantifying DNA Damage Foci in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Exponential growing cells were treated for 24 h with 5-azadC. During the last 12 h of this treatment cultures were grown in the presence or absence of DRB (10μM).
Alternatively, cells were treated with 5-azadC (15μM) for 6 h and DRB (30μM) was present during the last 3 h.
After treatments, cells were washed in PBS and incubated for 30s with ice cold 0.1% PBS containing Triton-X in order to pre-extract soluble protein. Then cells were fixed in 4% paraformaldehyde at room temperature for 10 min. Immunofluorescence of γ-H2AX (millipore), 53BP1 (Santa Cruz) and RAD51 (Santa Cruz) were carried out as previously described (21). Cells were photographed using a Zeiss LSM 7 duo confocal microscope.
The number of foci per cell was assessed using Image J software (NIH). Also, the percentage of cells with more than 10 or 20 foci were scored. At least 200 nuclei were counted on each slide.

Burgos-Morón E., Calderón-Montaño J.M., Pastor N., Höglund A., Ruiz-Castizo Á., Domínguez I., López-Lázaro M., Hajji N., Helleday T., Mateos S, & Orta M.L. (2018). The Cockayne syndrome protein B is involved in the repair of 5-AZA-2′-deoxycytidine-induced DNA lesions. Oncotarget, 9(80), 35069-35084.

+ Open protocol

+ Expand

Western Blot Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein lysates were electrophoresed on precast gels (Invitrogen) and transferred onto nitrocellulose membranes (GE Healthcare, Amersham, UK) by semi-dry blotting. Antibody binding was visualised using enhanced chemilumunescence (GE healthcare). Antibodies were obtained as follows: E1A (mouse, Becton Dickinson 554155), Ad5 (goat, Abcam Ab36851), RAD51 (rabbit, Santa-Cruz sc-8348), mcm3 (rabbit, Abcam Ab36851), Ku70 (goat, Santa-Cruz, sc-1486), (rabbit, Cell Signaling, 4895). All HRP-conjugated secondary antibodies were obtained from Dako (Ely, UK).

Tookman L.A., Browne A.K., Connell C.M., Bridge G., Ingemarsdotter C.K., Dowson S., Shibata A., Lockley M., Martin S.A, & McNeish I.A. (2015). RAD51 and BRCA2 enhance oncolytic adenovirus type 5 activity in ovarian cancer. Molecular cancer research : MCR, 14(1), 44-55.

+ Open protocol

+ Expand

PARP Inhibitor Screening Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Chemical compounds, including PARP inhibitors Olaparib (#S1060) and Rucaparib (#S1098), Novobiocin (#S2492), etoposide (#S1225), and PU-H71 (#S8039) were purchased from Selleckchem. Chemicals were dissolved in DMSO and kept in small aliquots at −80°C. The key sources of antibodies are: POLθ (Sigma #SAB1402530), γH2AX (Millipore #05–636), RAD51 (Santa Cruz Biotechnology, SCB, #sc-8349), CDK6 (SCB, #sc-7961), AKT1 (SCB #sc-5298), HSP70 (SCB #sc-24), HSP90 (SCB #sc-13119), and FANCF (Everest Biotech, #EB06112).

Zhou J., Gelot C., Pantelidou C., Li A., Yücel H., Davis R.E., Farkkila A., Kochupurakkal B., Syed A., Shapiro G.I., Tainer J.A., Blagg B.S., Ceccaldi R, & D’Andrea A.D. (2021). A first-in-class Polymerase Theta Inhibitor selectively targets Homologous-Recombination-Deficient Tumors. Nature cancer, 2(6), 598-610.

+ Open protocol

+ Expand

BRCA2 Protein Interactome Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

293T cells were transfected with pCDNA-HA-BRCA2 plasmids(i.e. wild-type HA-BRCA2, germline c.407delA HA-BRCA2del407 and putative reversion c.402_413delTCTAAATTCTTG HA-BRCA2REV) for 72 hrs, and lysed in lysis buffer (0.5% NP40, 25 mM Tris pH 7.5, 450 mM NaCl, 0.5 mM EDTA and proteinase inhibitors). The cell lysates were then incubated with anti-HA agarose beads (Sigma). After three washes with wash buffer (0.5% NP40, 25 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA), the beads were boiled in SDS sampling buffer, followed by western blotting with antibodies against HA (Santa Cruz), PALB2 (Novus Biologicals) and RAD51 (Santa Cruz).

Weigelt B., Comino-Méndez I., de Bruijn I., Tian L., Meisel J.L., García-Murillas I., Fribbens C., Cutts R., Martelotto L.G., Ng C.K., Lim R.S., Selenica P., Piscuoglio S., Aghajanian C., Norton L., Murali R., Hyman D.M., Borsu L., Arcila M.E., Konner J., Reis-Filho J.S., Greenberg R.A., Robson M.E, & Turner N.C. (2017). Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(21), 6708-6720.

+ Open protocol

+ Expand

Lung Cancer Cell Line Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols

NSCLC cell lines A549, HCC4006 and Calu-1 (three human lung adenocarcinoma cell lines), and SK-MES-1 and KLN205 (two human lung squamous carcinoma cell lines) were obtained from the Shanghai Institute for Biological Science (China). All cell lines were purchased between 2012 and 2015 and authenticated based on growth rate, morphology, and viability and were frequently confirmed to be mycoplasma free. The cells were cultured in RPMT 1640 media supplemented with 10% heat-inactivated fetal calf serum (FBS), L-glutamine and 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. The cells were maintained in a humidified incubator in 5% CO₂ at 37 °C.
Antibodies to various antigens were as follows: CHK1, PCHK1 (S296), PCHK1 (S317), PCHK1 (S345), CHK2, PCHK2 (T68), PCHK2 (S516), Cdc25A, PCdc25C (S216), H2AX, γH2AX (S139), PH3 (S10histone), ATM, PATM (S1981), DNA-PKCs, PDNA-PKCs (S2056), and GAPDH were from Cell Signaling. FANCL, FANCD2, BRCA2, PARP1, RAD51, caspase-3, cleaved caspanse-3, PARP and cleaved PARP from Santa Cruz. Gemcitabine was from Hanson Pharmaceutic (China), Cisplatin from Yangtze River Pharmaceutic (China), MK-8776 from Selleck Chemicals, CHK2 inhibitor II from Abcam. Gemcitabine was dissolved in PBS. Cisplatin, MK-8776 and CHK2 inhibitor II were dissolved in DMSO and used at the specified concentrations.

Dai C.H., Wang Y., Chen P., Jiang Q., Lan T., Li M.Y., Su J.Y., Wu Y, & Li J. (2017). Suppression of the FA pathway combined with CHK1 inhibitor hypersensitize lung cancer cells to gemcitabine. Scientific Reports, 7, 15031.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!