Gamma h2ax

Manufactured by Cell Signaling Technology

Sourced in United Kingdom, United States

Gamma-H2AX is a molecular marker that is phosphorylated on serine 139 of the histone H2AX in response to DNA double-strand breaks. It serves as a sensitive indicator of DNA damage.

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Lab products found in correlation

7 protocols using gamma h2ax

Real-time PCR Gene Expression Assay

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All the real time PCR Taqman gene assays were purchased from Life Technologies (MSH2: Hs00953523_m1; MLH1: Hs00179866_m1; MSH6: Hs00264721_m1; PMS2: HS00241053_m1; Rad51: Hs00153418_m1; Chk1: Hs00967506_m1). The antibodies to Caspase3 (#9662), Rad51 (#8875), MLH1 (#3515), Chk1 (#2360), Gamma H2A.X (#9718), MSH2 (#2850), Topoisomerase IIα (D10G9) (#12286) were purchased from Cell Signaling; PMS2 (#2251.00.02), MSH6 (#2203.00.02), were purchased from Sdix, Alpha tubulin - Abcam (ab15246); Lamin A/C -Cell Signaling (#2032) and beta-Actin (#sc-81178) was purchased from Santa Cruz Biotechnology. The secondary antibodies for Western Blotting were purchased form LI-COR and for immunofluorescence were purchased from Invitrogen (#A11008).

Ranganathan P., Kashyap T., Yu X., Meng X., Lai T.H., McNeil B., Bhatnagar B., Shacham S., Kauffman M., Dorrance A.M., Blum W., Sampath D., Landesman Y, & Garzon R. (2016). XPO1 Inhibition Using Selinexor Synergizes With Chemotherapy in Acute Myeloid Leukemia (AML) by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(24), 6142-6152.

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Protein Extraction and Western Blot Analysis

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Ventricles were collected and lysed in RIPA buffer (Millipore Sigma) with the addition of a complete protease inhibitor cocktail (Roche). Protein concentration was determined using Pierce BCA protein assay kit (Pierce Biotechnology), with three biological replicates. Following separation via SDS-PAGE gels, proteins were transferred to nitrocellulose membranes (Bio-Rad), blocked in 5% skim milk/TBS, and incubated with primary antibodies: Gamma H2AX (Cell signaling 9718, 1:1,000); p-ATM (Santa Cruz Biotechnology sc-47739, 1:1,000); ATM (Genetex GTX70103, 1:1,000); p-CHK2 (Abcam ab59408, 1:1,000); CHK2 (Cell signaling 2662T, 1:1,000); p-CHK1 (Cell signaling 2348S, 1:1,000); CHK1 (Cell signaling 2360S, 1:1,000); GAPDH (Millipore AB2302, 1:6,000). Horseradish peroxidase-conjugated peroxidase anti-mouse, anti-rabbit, or anti-goat antibodies were used as secondary antibodies (ImmunoResearch: 115–035-166, 111–035-144, 703–035-155, 705–035-147; 1:25,000–1:50,000). The membranes were exposed using Licor Odyssey Fc system and quantified by Image Studio Lite v.5.2 software.

Ali S.R., Nguyen N.U., Menendez-Montes I., Hsu C.C., Elhelaly W., Lam N.T., Li S., Elnwasany A., Nakada Y., Thet S., Foo R.S, & Sadek H.A. (2024). Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage. The journal of cardiovascular aging, 4(1), 11.

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Quantification of DNA Damage Markers

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ATM (ab36810, ABCAM, UK), gamma-H2AX (2577, Cell Signaling, MA, USA), Ki67 (sc7846, SantaCruz Biotech, CA, USA), RAD51 (ab88572, ABCAM, UK) DNA-PK (sc390698, SantaCruz Biotech, CA, USA) and 53BP1 (ab172580, ABCAM, UK) were detected according to manufacturers' protocols. Hoechst 33342 staining was performed, and then cells were observed through a fluorescence microscope (Leica Italia, Italy). The percentage of ATM-, gamma-H2AX-, Ki67-, RAD51-, DNA-PK and 53BP1-positive cells was calculated by counting at least 500 cells in different microscope fields.

Alessio N., Del Gaudio S., Capasso S., Di Bernardo G., Cappabianca S., Cipollaro M., Peluso G, & Galderisi U. (2014). Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process. Oncotarget, 6(10), 8155-8166.

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Protein Isolation and Analysis Protocol

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Total protein (~20 μg) was isolated from cells following 72 h treatment or vehicle control (0.02% DMSO or ethanol) for protein analysis as previously describe [40 (link),41 (link)]. Tris-glycine gels were used for all analysis except for BRCA1, ATM and the respective loading control where Tris-acetate gels were used to accommodate higher molecular weights of these proteins. The following antibodies were used: Actin (#4967), ATM (#2873), BRCA1 (#9010), gamma-H2AX (#80312), Rad51 (#8875), total-H2AX (#7631), p62 (#2947) and LC3BII (#2775) were from Cell Signaling (Danvers, MA, USA); CYP1B1 (ab185954) was from Abcam; β-tubulin (T7816) was from Sigma; DNA/RNA damage antibody (SMC-155) was from StressMarq (Victoria, BC, Canada) and Actin (#47778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Bakewell S., Conde I., Fallah Y., McCoy M., Jin L, & Shajahan-Haq A.N. (2020). Inhibition of DNA Repair Pathways and Induction of ROS Are Potential Mechanisms of Action of the Small Molecule Inhibitor BOLD-100 in Breast Cancer. Cancers, 12(9), 2647.

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Fluorescence Analysis of DNA Damage

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Briefly, after 0 and 1440min of 169MHz RF exposure, the cells were fixed in 4% formaldehyde (Sigma-Aldrich) solution from 15mins at RT (20–23°C). Then, we permeabilized the cells with 0.3% Triton-X100 (Roche, Basel, Switzerland) on ice for 5 min and then added a 5% FBS solution in PBS and 0.1% Triton-X100 for 1 h at RT (Blocking Solution). Subsequently, the antibodies ATM (1:1000, ab36810, ABCAM, Cambridge, UK) and gamma-H2AX (1:600, #2577, Cell Signaling Technology, Danvers, MA, USA) were detected according to manufacturers’ protocols. The FITC-conjugated secondary antibody, goat anti-rabbit (1:400, Gtx-Rb-003D488) or TRITC-conjugates secondary antibody goat anti-mouse (1:400, Gtx-Mu-003D594) were obtained from ImmunoReagents (Raleigh, NC, USA). All the antibodies were diluted in blocking solution. Nuclear staining was performed using DAPI mounting medium (ab104139, ABCAM) and micrographs were captured under a fluorescence microscope (Leica DM2000,-DMC5400, Leica, Wetzlar, Germany). The percentage of ATM- and gamma-H2AX-cells was calculated by counting at least 300 cells in different microscopic fields.

Alessio N., Santoro E., Squillaro T., Aprile D., Briccola M., Giubbini P., Marchesani R., Muoio M.R, & Lamberti M. (2019). Low-Level Radiofrequency Exposure Does Not Induce Changes in MSC Biology: An in vitro Study for the Prevention of NIR-Related Damage. Stem Cells and Cloning : Advances and Applications, 12, 49-59.

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Real-time PCR and Western Blot Assays

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Real-time PCR Taqman gene probes were purchased from Life Technologies (Carlsbad, CA, USA) (MSH2: Hs00953523_m1; MLH1: Hs00179866_m1; MSH6: Hs00264721_m1; PMS2: HS00241053_m1; Rad51: Hs00153418_m1; CHEK1: Hs00967506_m1). The antibodies for PARP-1 (#9542), Caspase-3 (#9662), Rad51 (#8875), MLH1 (#3515), CHEK1 (#2360), Gamma H2A.X (#9718), MSH2 (#2850) were purchased from Cell Signaling (Danvers, MA, USA); PMS2 (#2251.00.02) and MSH6 (#2203.00.02) antibodies were purchased from Sdix (Newark, DE, USA). Antibodies targeting XPO1 (#sc-5595) and beta-actin (#sc-81178) were purchased from Santa Cruz Biotechnology. The secondary antibodies for western blotting were purchased form LI-COR (Lincoln, NE, USA) and the secondary antibodies for immunofluorescence were purchased from Life Technologies (#A11008).

Kashyap T., Argueta C., Unger T., Klebanov B., Debler S., Senapedis W., Crochiere M.L., Lee M.S., Kauffman M., Shacham S, & Landesman Y. (2018). Selinexor reduces the expression of DNA damage repair proteins and sensitizes cancer cells to DNA damaging agents. Oncotarget, 9(56), 30773-30786.

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Gemcitabine-KPT-330 Combination Treatment

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MiaPaCa-2 cells were seeded on coverslips in 6-well plates at a density of 500,000 cells/well. The next day, cells were treated with gemcitabine (5 µM) or DMSO for 30 hours, followed by exposure to KPT-330 (1 µM) for the next 6 hours. Cells were fixed with methanol and stained with gamma H2A.X (Cell Signaling) antibody. Nuclei were stained with DAPI.

Kazim S., Malafa M.P., Coppola D., Husain K., Zibadi S., Kashyap T., Crochiere M., Landesman Y., Rashal T., Sullivan D.M, & Mahipal A. (2015). Selective nuclear export inhibitor KPT-330 enhances the antitumor activity of gemcitabine in human pancreatic cancer. Molecular cancer therapeutics, 14(7), 1570-1581.

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