Rad50

Manufactured by Cell Signaling Technology

Sourced in United States

Rad50 is a laboratory instrument used for the detection and analysis of the Rad50 protein, which plays a crucial role in DNA double-strand break repair. The core function of Rad50 is to facilitate the recognition and processing of DNA damage, a fundamental process in maintaining genomic integrity.

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Lab products found in correlation

Close spelling variants of this product

Anti-Rad50 (5 citations) P-RAD50 (2 citations) Anti-phospho-Rad50 (Ser635) (2 citations) Rabbit anti-RAD50 (2 citations)

23 protocols using rad50

Identification of rLOX-PP Interactors

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Empty vector and rLOX-PP expressing PC3 and DU145 cells were cultured, extracted into non-denaturing RIPA cell lysis buffer and incubated with agarose bead-immobilized Non-immune IgG or anti-c-Myc tag IgG overnight at 4°C to pull down rLOX-PP. Non-immune IgG (#31903, Thermo Scientific, Waltham, MA) was coupled to agarose using AminoLink Immobilization Plus Trial kit (#20394, Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. For pull-down assays, beads were washed, and then eluted according to kit instructions (ProFound c-Myc Tag IP/Co-IP Kit; Thermo Scientific, Waltham, MA). Eluted samples were analyzed by Western blotting on denaturing SDS PAGE probed with an anti-ATM, anti-ATR, Rad50, anti-NBS1 and anti-MRE11 antibodies (Cell Signaling, Danvers, MA) and with anti-rLOX-PP antibody (61 (link)). Aliquots (5%) of initial extracts taken before the immunoprecipitation were analyzed by Western blot on the same gels.

Bais M.V., Ozdener G.B., Sonenshein G.E, & Trackman P.C. (2014). Effects of Tumor Suppressor Lysyl Oxidase Propeptide on Prostate Cancer Xenograft Growth and Its Direct Interactions with DNA Repair Pathways. Oncogene, 34(15), 1928-1937.

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Whole-cell Protein Extraction and Western Blotting

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Whole-cell protein extractions was acquired using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Cat# 89901), supplemented with protease inhibitor (Thermo Fisher Scientific, Cat#78430). The concentration of protein was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Cat# J63283.QA) before mixed with protein loading buffer (3×). Then the protein samples were separated by 10% SDS-PAGE, and the detailed procedures conducted as previously described[20 (link)].
Antibodies including α-Tubulin, GAPDH, Cyclin D1, P21, PCNA, E2F1, CDK2, CDK4, CDK6, RB1, JAG1, JAG2, Caspase 8, BAX, XIAP, BCL-2L12, ATR, ATM, Rad50, Mre11, PIK3C3, LC3A/B, and ULK1 were purchased from Cell Signaling Technology (Danvers, MA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States).

Liu D., Shi Y., Chen H., Nisar M.A., Jabara N., Langwinski N., Mattson S., Nagaoka K., Bai X., Lu S, & Huang C.K. (2023). Molecular profiling reveals potential targets in cholangiocarcinoma. World Journal of Gastroenterology, 29(25), 4053-4071.

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Resveratrol-Induced DNA Damage Response

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Cells were cultured and treated with resveratrol for different time periods and different concentrations. Next, cells were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mg/mL PMSF, 2 mg/mL aprotinin, 2 mM leupeptin, and 1 mg/mL pepstatin) and protein concentration was determined using the Bradford assay. Protein extracts were resolved by SDS–PAGE (60 mg per lane) on a 10% polyacrylamide gel and transferred into immobilon membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, membranes were incubated with 1:1000 dilutions of primary antibodies. We used antibodies against p-ATM, p-BRCA1 and γ-H2AX (Cell Signaling Technology, Inc., Danvers, MA, USA) as markers of DNA damage response; active-caspase 3 and cleaved PARP (Cell Signaling Technology, Inc., Danvers, MA, USA) to determine apoptosis marker; and finally, Rad50, Mre11, p-p95/NBS1, DNA-PKcs, and KU80 (Cell Signaling Technology, Inc, Danvers, MA, USA) as markers of DNA repair; Tubulin (Calbiochem, Darmstadt, Germany) was used as loading control.

Jara P., Spies J., Cárcamo C., Arancibia Y., Vargas G., Martin C., Salas M., Otth C, & Zambrano A. (2017). The Effect of Resveratrol on Cell Viability in the Burkitt’s Lymphoma Cell Line Ramos. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(1), 0014.

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DNA Damage Response Protein Analysis

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Western blot was performed as described previously (19 (link), 20 (link)), using pATM (Ser1981) (D6H9), MRE11, RAD50, NBS1, PARP-1, pAKT pCHK2 (Thr68) (C13C1), γH2AX (Cell Signaling, Danvers, MA), and β-Actin (8H10D10) antibodies (Cell Signaling). Appropriate horseradish peroxide-conjugated secondary antibodies (Cell Signaling) were used, and proteins were detected with the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). The blotted membranes were stripped and re-probed with ATM (D2E2), AKT, and CHK2 (D9C6) antibodies (Cell Signaling). Protein bands were captured and quantified by Chemi Doc^TM MP Imaging System (Bio-Rad).

Zhao J., Nguyen L.N., Nguyen L.N., Dang X., Cao D., Khanal S., Schank M., Thakuri B.K., Ogbu S.C., Morrison Z.D., Wu X.Y., Li Z., Zou Y., El Gazzar M., Ning S., Wang L., Moorman J.P, & Yao Z.Q. (2019). ATM Deficiency Accelerates DNA Damage, Telomere Erosion, and Premature T Cell Aging in HIV-Infected Individuals on Antiretroviral Therapy. Frontiers in Immunology, 10, 2531.

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Whole-Cell Protein Extraction and Western Blot

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Whole-cell lysates were harvested using RIPA buffer (Cell Signaling Technology) containing protease inhibitor cocktail, the PhosSTOP phosphatase inhibitors and PMSF (Roche, Basel, Switzerland). 30 μg of lysate protein were then fractionated in SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). After blocking antigen, the membranes were incubated with primary antibodies and the corresponding secondary antibodies. The primary antibody used in this assay included Rab1A (Santa Cruz, USA) and GAPDH, S6K1, P-S6K1(T398), E-cadherin, N-cadherin, Vimentin, ZO-1, H2AX, γ-H2AX, Rad50, Mer11, P-NBS1(Ser343), P-ATM(Ser1981), Ku80, DNA-PKcs, β-catenin, GSK-3β, P-GSK-3β(Ser9) (Cell Signaling Technology) and Rad51 (Proteintech Group, USA). Finally, proteins were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Waltham, MA, USA).

Yang X.Z., Chen X.M., Zeng L.S., Deng J., Ma L., Jin C., Wang R., Wang M.H., Wen Y.F., Wu X.L., Wang H.Y, & Cui S.Z. (2020). Rab1A promotes cancer metastasis and radioresistance through activating GSK-3β/Wnt/β-catenin signaling in nasopharyngeal carcinoma. Aging (Albany NY), 12(20), 20380-20395.

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Antibody-based Analysis of DNA Damage Repair

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Antibodies against the following proteins were used for immunofluorescence (IF) and Western blotting (WB): γH2AX (05–636 JBW301; Upstate Biotechnology), CENPF (ab5; Abcam), RPA (NA-18, Ab-2; Calbiochem for IF, LS-C38952; LifeSpan BioSciences for WB), pRPA S4/8 (A300-245A; Bethyl Laboratories), p DNA-PKcs S2056 (ab18192), DNA-PKcs (12311; Cell Signaling), Ku80 (2180 (C48E7); Cell Signaling), pKAP-1 S824 (A300-767A; Bethyl Laboratories), KAP-1 (ab3831; Abcam), MRE11 (12D7, GTX70212; GeneTex), NBS1 (PC269, Ab-1; Oncogene), RAD50 (3427; Cell Signaling), pATM S1981 (2152–1; Epitomics), ATM (GTX70103 2C1; GeneTex), CtIP (A300-488A; Bethyl Laboratories), and β-tubulin (ab21058; Abcam). Ku70, XLF, XRCC4, and Ligase IV antibodies were kind gifts from Dr. Penny Jeggo. Immunoblotting was performed as described previously [17 (link)].

Nakajima N.I., Hagiwara Y., Oike T., Okayasu R., Murakami T., Nakano T, & Shibata A. (2015). Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair. PLoS ONE, 10(3), e0122582.

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Comprehensive Antibody Panel for DNA Repair

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Mouse antibodies used were against Flag M2, α- and β-tubulin (Sigma), BRCA1, γH2AX (Millipore), LIG4, GFP and c-myc (Santa Cruz), REV7 (BD Transduction Libratory); rabbit antibodies were against PARP1, H2AX, FEN1, RAD50 (Cell Signalling), Histone H3, BLM, DNA2, DYNLL1 (Abcam), MRE11 (GeneTex), ATMIN (Millipore), EXO1 (Sigma), PTIP, 53BP1and Rad51 (Santa Cruz), RPA32 Phosphor-S4/S8 (Bethyl); goat antibodies were against NBN (Santa Cruz) and rat antibody against RPA2 (Cell Signaling).

He Y.J., Meghani K., Caron M.C., Yang C., Ronato D.A., Bian J., Sharma A., Moore J., Niraj J., Detappe A., Doench J.G., Legube G., Root D.E., D’Andrea A.D., Drané P., De S., Konstantinopoulos P., Masson J.Y, & Chowdhury D. (2018). DYNLL1 binds MRE11 to limit DNA end resection in BRCA1-deficient cells. Nature, 563(7732), 522-526.

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Protein Extraction and Western Blot Analysis

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Total cellular proteins and nuclear proteins were extracted using lysis buffer (Pierce, Rockford, IL, USA). The method applied has been described in detail in our previous study.9 Primary antibodies, including phospho‐eIF2α, phospho‐ATM, Bcl‐2, cleaved‐caspase3, DNA‐PK, Rad50, Nbs1, Mre11, Bcl‐xL, cleaved poly(ADP‐ribose) polymerase (PARP), phospho‐p65, p65, cyclin B, phosphor‐Cdc2 (Try15), β‐actin, histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), IRE‐1, ATF‐6,HIF‐1α and PERK (1:1000; Abcam), at 4°C overnight. Secondary antibodies, anti‐mouse or anti‐rabbit IgG antibody (1:1000 dilution; Cell Signaling Technology), were added and incubated at room temperature for 2 h. Target proteins on PVDF membranes were visualized with LumiGLO (Cell Signaling Technology) and captured using a DNR BioImaging System (DNR, Israel).

Qiao Q., Sun C., Han C., Han N., Zhang M, & Li G. (2017). Endoplasmic reticulum stress pathway PERK‐eIF2α confers radioresistance in oropharyngeal carcinoma by activating NF‐κB. Cancer Science, 108(7), 1421-1431.

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TNBC Cell Line Maintenance and Treatment

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Three TNBC cell lines (Hs578t, MDA-MB-231, and SUM159) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Grand Island, NY) with 10% fetal bovine serum (Thermos Scientific Hyclone, Rockford, IL) in a humidified 5% CO₂ incubator at 37°C. Chloroquine diphosphate salt (Sigma-Aldrich, St. Louis, MO) and carboplatin (Teva Pharmaceuticals, Sellersville, PA) were dissolved in cell culture-grade water at the concentration of 10 mM. CD marker antibodies (BD Biosciences, San Jose, California), MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific, Waltham, MA), Cell Proliferation Reagent WST-1 (Clontech, Mountain View, CA) were acquired. Cytochrome C, LC-3B, p62, NQO1, Rad50, PARP and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA). Rad51, β-tubulin, and Tom20 antibodies were purchased from Santa Cruz Biotechnology Inc. Dallas, TX. γ-H₂AX (EMD Millipore, Billerica, MA) and bcl-2 antibodies (BioLegend, San Diego, CA), were purchased from their respective companies.

Liang D.H., Choi D.S., Ensor J.E., Kaipparettu B.A., Bass B.L, & Chang J.C. (2016). The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair. Cancer letters, 376(2), 249-258.

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Western Blot Analysis of DNA Damage Signaling

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Protein concentrations were determined using the BCA kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Protein lysates (20-100 μg) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies: Phospho-ATM (Ser1981) (D6H9) from rabbit (1:500, 5883, Cell Signaling, Danvers, MA), Phospho-BRCA1 (Ser1524) from rabbit (1:500, 9009, Cell Signaling), DNA-PK from rabbit (1:500, 4602, Cell Signaling), Mre11 (31H4) from rabbit (1:500, 4847, Cell Signaling) and Rad50 from rabbit (1:500, 3427, Cell Signaling). Membranes were then incubated with a 1:5,000 dilution of a peroxidase conjugated corresponding secondary antibody. Equal loading of the protein samples was confirmed by parallel western blots for β-actin (1:5,000, ab822750; Abcam). Detection was performed using the ECL-Enhanced Chemiluminescence Detection System (GE Healthcare Biosciences, Pittsburgh, PA) according to the manufacturer's instructions. Blots were visualized by autoradiography.

Ambati S.R., Shieh J.H., Pera B., Lopes E.C., Chaudhry A., Wong E.W., Saxena A., Su T.L, & Moore M.A. (2016). BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models. Oncotarget, 7(28), 43062-43075.

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