γ h2ax

Manufactured by Abcam

Sourced in United States, United Kingdom, China

γ-H2AX is a phosphorylated variant of the H2AX histone protein. It is a marker of DNA double-strand breaks and is widely used in the study of the DNA damage response.

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Lab products found in correlation

Cleaved caspase 3, by Cell Signaling Technology (18 mentions) Pvdf membrane, by Merck Group (12 mentions) β actin, by Abcam (10 mentions) Immuno block reagent, by Bio SB (10 mentions) Rad51, by Abcam (10 mentions) Caspase 3, by Cell Signaling Technology (8 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions) Bca protein assay kit, by Thermo Fisher Scientific (7 mentions) Gapdh, by Cell Signaling Technology (6 mentions) β actin, by Santa Cruz Biotechnology (6 mentions) Bcl 2, by Abcam (6 mentions) β actin, by Merck Group (6 mentions) α actinin, by Santa Cruz Biotechnology (5 mentions) β actin, by Cell Signaling Technology (5 mentions) Flag tag (flag), by Merck Group (5 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (5 mentions) Triton x 100, by Merck Group (5 mentions) Cleaved parp, by Cell Signaling Technology (5 mentions) Gapdh, by Abcam (5 mentions) Gapdh, by Santa Cruz Biotechnology (5 mentions)

189 protocols using γ h2ax

DNA Fiber Analysis and Western Blot Protocols

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Antibodies used for DNA fiber analysis were: anti-BrdU (BD Biosciences, Clone B44, 347580), anti-BrdU (Abcam, ab6326), Alexa-Fluor 568 conjugated anti-mouse secondary (Thermo Scientific, A11004), Alexa-Fluor 488 conjugated anti-rat secondary (Thermo Scientific, A11006).
Antibodies used for western blot analysis were: Tubulin (Millipore Sigma, clone DM1A, 05–829), NS1 (2C9b monoclonal antibody), phospho-RPA32 (Cell Signaling, 83745), γH2AX (Abcam, ab11174), phospho-MCM2 (Thermo Fisher, PA5-106187), HRP-conjugated anti-mouse secondary (Cell Signaling, 7076S), HRP conjugated anti-rabbit secondary (Cell Signaling, 7074S).
Antibodies used for immunofluorescence analysis were: NS1 (2C9b monoclonal antibody), RPA32 (Cell Signaling, 2208S), phospho-RPA32 (Cell Signaling, 83745S), phospho-MCM2 (Thermo Fisher, PA5-106187), γH2AX (Abcam, ab11174), Alexa-Fluor 568 conjugated anti-mouse secondary (Thermo Scientific, A11004), Alexa Fluor 488 conjugated anti-rat secondary (Thermo Scientific, A11006), Alexa-Fluor 647 conjugated anti-rabbit secondary (Thermo Scientific, A21245).
Antibodies used for ChIP-qPCR analysis were: RPA (Cell Signaling, 2208S).

Haubold M.K., Aquino J.N., Rubin S.R., Jones I.K., Larsen C.I., Pham E, & Majumder K. (2023). Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion. PLOS Pathogens, 19(5), e1011203.

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Western Blot Analysis of Cellular Proteins

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The cell was lysed with RIPA (radioimmunoprecipitation assay) lysis buffer (150 mM NaCl, 0.1% SDS, 100 mM Tris-HCl of pH 8.0, and 1% Triton X-100) and then centrifuged at 12,000 r.p.m., 4°C for 15 min. The supernatant of each sample was mixed with protein loading dye, equally loaded into and subsequently separated by SDS-PAGE, transferred to a polyvinylidine difluoride membrane (Millipore, USA), and finally incubated with primary antibodies against ATF3, p21, p53, cyclin D1, α-actinin (ACTN), histone H3 phosphorylation (at serine 10), HSP90α/β (Santa Cruz Biotechnology, USA), PARP, LC3B (light chain 3B) (Cell Signaling, USA), and γH2A.x (phosphorylated form of H2A.x at serine 139) (Epitomics, USA) at 4°C overnight, followed by the secondary antibody incubation and ECL detection.

Lin C.K., Liu S.T., Chang C.C, & Huang S.M. (2019). Regulatory mechanisms of fluvastatin and lovastatin for the p21 induction in human cervical cancer HeLa cells. PLoS ONE, 14(4), e0214408.

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Immunofluorescent Quantification of DNA Double-Strand Breaks

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DSBs represent an important ionizing radiation-induced lesion. The rapid phosphorylation of histone H2AX at serine 139 is a sensitive marker for DNA DSBs induced by ionizing radiation, which can later be detected by immunofluorescence.
The treated TE-1 cells were placed in 24−well plates with circular slides were fixed in 4% paraformaldehyde, permeabilized with 1% Triton X-100 for 10 min at room temperature, and then blocked with dilute 1% BSA (Solarbio, Beijing, China) for 1 h. The cells were incubated at 4°C overnight with the primary antibody γ-H2AX (Epitomics, Burlingame, CA, USA), which was diluted 1:1000 with 1% BSA. PBS was used to wash away the primary antibody. The cells were incubated with the secondary antibody at 37°C for 1 h, following which they were placed on coverslips and counterstained with 4-6-diamidino-2-phenylindole (Invitrogen) to mark the nuclei. Immunofluorescence staining was examined using a confocal microscope (Olympus Corporation) and then the mean number of γ-H2AX foci per cell (foci/cell) were counted.

Hu Y., Li Q., Yi K., Yang C., Lei Q., Wang G., Wang Q, & Xu X. (2022). HuR Affects the Radiosensitivity of Esophageal Cancer by Regulating the EMT-Related Protein Snail. Frontiers in Oncology, 12, 883444.

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Western Blot Analysis of Cellular Signaling

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Western blot analyses were performed as previously described 28 (link). In brief, cells were lysed in whole-cell lysate buffer containing 1% phosphatase inhibitor cocktail and NPC tumor tissues were lysed in RIPA buffer (Beyotime, Jiangsu, China) with 1% phosphatase inhibitor cocktail. Lysates containing 20-30 µg protein were loaded onto 8% or 12% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis (SDS-PAGE) and the separated proteins transferred to poly vinylidene fluoride (PVDF) membranes (Pall, NY, USA). After blocking with 5% fat free milk for 1 h in Tris-buffered saline (TBS), the membranes were incubated with the primary antibody overnight at 4°C and then with the peroxidase labelled secondary antibody (agilent, CA, USA) for 1 h on the next day. WB bands were visualized with an enhanced chemiluminescence kit (EMD Millipore, MA, USA). The primary antibodies used were γH2AX (1:2000 dilution, Epitomics, Burlingame, CA, USA) and monoclonal anti-cleaved PARP-1, β-actin, p-AKT, p-mTOR, SQSTM1/p62, LC-3, P-P65 (1:2000 dilution, Cell Signaling Technology, Danvers, MA, USA).

Xu W., Wei Q., Han M., Zhou B., Wang H., Zhang J., Wang Q., Sun J., Feng L., Wang S., Ye Y., Wang X., Zhou J, & Jin H. (2018). CCL2-SQSTM1 positive feedback loop suppresses autophagy to promote chemoresistance in gastric cancer. International Journal of Biological Sciences, 14(9), 1054-1066.

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Cell Signaling Pathway Analysis

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Cell lysates were prepared in lysis buffer (100 mM Tris-HCl of pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100) at 4° C. The extracts were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore, USA) and detected using antibodies against PARP, LC3 (Cell Signaling, USA), SRSF3, Slu7, p53, p21, cyclin D1, ATF3, COX-2, α-actinin (ACTN), HuR (Santa Cruz Biotechnology, USA), and γ−H2AX (Epitomics, USA).

Chang Y.L., Liu S.T., Wang Y.W., Lin W.S, & Huang S.M. (2018). Amiodarone promotes cancer cell death through elevated truncated SRSF3 and downregulation of miR-224. Oncotarget, 9(17), 13390-13406.

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Western Blot Analysis of Cell Lysates

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The cell lysates were prepared in lysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS and 1% Triton X-100) at 4°C. The extracts were separated by SDS-PAGE, transferred onto a polyvinylidine difluoride membrane (Millipore, MA, USA) and detected using antibodies against PARP, LC3 (Cell Signaling Technology, MA, USA), α-actinin (ACTN), p53, p21, MLC-2v, H3P, HuR, Twist, CACNb1 (Santa Cruz Biotechnology, CA, USA), γH2A.X, myogenin (Epitomics, CA, USA) and hemagglutinin (HA) (3F10, Hoffmann-La Roche, Basel, Switzerland).

Liu S.T., Huang S.M., Ho C.L., Yen L.C., Huang C.J., Lin W.S, & Chan J.Y. (2015). The regulatory mechanisms of myogenin expression in doxorubicin-treated rat cardiomyocytes. Oncotarget, 6(35), 37443-37457.

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Pharmacodynamics of DNA Damage Markers

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300-600 mm³ HT29 xenograft tumors were used in the pharmacodynamics studies. Six animals per group were treated with vehicle, AZD7762 25 mg/kg, ip, on Day 1 and Day 2, TH-302 150 mg/kg, ip on Day 1 or the combination of AZD7762 and TH-302. In the combination treatment, two dosing sequences were used: TAA and ATA (as above). Tumors were harvested on Day 3 (which was 24 h after the second AZD7762 treatment), and fixed in 10% neutral buffered formalin and embedded in paraffin. 5 μm thick paraffin sections were cut and adhered to poly-_L-lysine-coated glass slides.
After deparaffinization and rehydration of the slides, antigen was retrieved. Endogenous peroxidase was quenched by Peroxidaze 1 and non-specific binding was blocked by Background Sniper (both Biocare Medical). Slides were incubated with rabbit monoclonal anti γH2AX (Epitomics, 1:3000), or rabbit polyclonal anti-phospho Chk1 (phospho S345, Chk1-S345, Abcam, 1:25) or Caspase 3 (Cell Signaling Technology, 1:300) for 1 h at RT followed by secondary HRP-conjugated anti rabbit IgG (Epitomics).

Meng F., Bhupathi D., Sun J.D., Liu Q., Ahluwalia D., Wang Y., Matteucci M.D, & Hart C.P. (2015). Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition. BMC Cancer, 15, 422.

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Western Blot Analysis of Epithelial-Mesenchymal Transition Markers

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The cells were washed twice with cold phosphate-buffered saline (PBS) and lysed on ice in RIPA buffer with 1% PMSF (KeyGen, Nanjin, China). The protein lysates were resolved on 10% SDS polyacrylamide gels, transferred to PVDF membranes and blocked in 0.1% Tween20 and 5% skim milk protein in Tris-buffered saline. The membrane was incubated with primary antibodies (E-cadherin, Vimentin, CD133, Sox2, p21, PTEN, AKT, p-AKT and γ-H2AX purchased from Epitomics company) followed by incubation with HRP-labelled rabbit IgG, and the proteins were detected via chemiluminescence. The membrane was then washed and visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The signals were detected with enhanced chemiluminescence (KeyGen, Nanjin, China).

Zheng L., Zhang Y., Liu Y., Zhou M., Lu Y., Yuan L., Zhang C., Hong M., Wang S, & Li X. (2015). MiR-106b induces cell radioresistance via the PTEN/PI3K/AKT pathways and p21 in colorectal cancer. Journal of Translational Medicine, 13, 252.

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Antibodies and Inhibitors for Cancer Research

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Antibodies used in this study are as follows: tubulin (#T5168) and Flag (#F3165) were purchased from Sigma-Aldrich (St. Louis, MO); PARP1 (#sc-7150) for Western blotting from Santa Cruz Biotechnology (Santa Cruz, CA); PARP1 (#9532) for immunoprecipitation (IP), MET (#8198), phosphorylated MET (#3077), phosphorylated EGFR for IHC (#3777) and EGFR (#4267) from Cell Signaling Technology (Danvers, MA); and phosphorylated EGFR for Western blotting (#5650), Ki67 (#21700), cleaved caspase 3 (#2302), γ-H2AX (#140498), and PAR (#14460) from Abcam (Cambridge, MA); 8-hydroxy-2′-deoxy guanosine (8-OHdG) from Genox Corporation (Baltimore, MD). The mouse phospho-Y907-PARP1 antibody was generated against a phosphorylated synthetic peptide (ADMVSKSAN-Yp-CHTSQGD) at China Medical University as described previously (13 (link)). The following inhibitors were used in this study: MET kinase inhibitor crizotinib (#C-7900) was purchased from LC Laboratories (Woburn, MA); EGFR inhibitor gefitinib (#S1025) and erlotinib (#S1023) from Selleck Chemicals (Houston, TX); PARP inhibitors ABT-888 (veliparib, #CT-A888) and AG014699 (rucaparib, #CT-AG01) from ChemieTek (Indianapolis, IN). Hydrogen peroxide (#216763) and sodium arsenite solution (#35000) were obtained from Sigma-Aldrich.

Dong Q., Du Y., Li H., Liu C., Wei Y., Chen M.K., Zhao X., Chu Y.Y., Qiu Y., Qin L., Yamaguchi H, & Hung M.C. (2018). EGFR and c-MET cooperate to enhance resistance to PARP inhibitors in hepatocellular carcinoma. Cancer research, 79(4), 819-829.

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Immunostaining Protocol for γ-H2AX and pH3

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Cells grown on 24-well plates were fixed for 15 min in 4% (w/v) para-formaldehyde (PFA)/PBS after treatment and then permeabilized for 15 min in 0.25% (v/v) Triton X-100/PBS. After fixation and permeabilization, cells were washed three times in PBS and then blocked with goat serum for 1 h. Cells were incubated with antibodies against γ-H2AX (Abcam), pH3 (Cell signaling technology) as required for 60 min, followed by three times wash with PBS and a 60 min incubation with goat anti-mouse cy3 or goat anti-rabbit FITC secondary antibody. Fluorescence images were taken under an Olympus fluorescent microscope.

Chen S., Chen X., Xie G., He Y., Yan D., Zheng D., Li S., Fu X., Li Y., Pang X., Hu Z., Li H., Tan W, & Li J. (2016). Cdc6 contributes to cisplatin-resistance by activation of ATR-Chk1 pathway in bladder cancer cells. Oncotarget, 7(26), 40362-40376.

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