Anti γh2ax 05 636

Manufactured by Merck Group

Sourced in United States

Anti-γH2AX (05-636) is a laboratory reagent that detects the presence of phosphorylated histone H2AX, a marker of DNA double-strand breaks. It is a primary antibody that binds to the γH2AX phosphorylated form of the H2AX histone protein.

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Lab products found in correlation

Cytospin 4 centrifuge, by Thermo Fisher Scientific (2 mentions) Alexa 568, by Thermo Fisher Scientific (2 mentions) Axiocam mrm ccd camera, by Zeiss (2 mentions) Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Poly d lysine coated culture slides, by BD (2 mentions) Axioimager m2 microscope system, by Zeiss (2 mentions) Plan apochromat 63 na 1.40 objective, by Zeiss (2 mentions) Axiovision software, by Zeiss (2 mentions) Anti phospho histone h3, by Merck Group (2 mentions) Vectashield mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (2 mentions) Anti β actin a1978, by Merck Group (1 mentions) Anti ha h9658, by Merck Group (1 mentions) Anti ub p4d1, by Santa Cruz Biotechnology (1 mentions) Anti flag f1804, by Merck Group (1 mentions) Anti 53bp1 a300 272a, by Fortis Life Sciences (1 mentions) Ha flag usp13, by Addgene (1 mentions) Pgex 4t 2 vector, by Takara Bio (1 mentions) Anti brca1 d9, by Santa Cruz Biotechnology (1 mentions) Anti mdc1, by Merck Group (1 mentions) Annexin 5 staining kit, by Roche (1 mentions)

13 protocols using anti γh2ax 05 636

Characterization of USP13 Regulation

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HA-FLAG-USP13 was purchased from Addgene (Plasmid #22568, provided by Dr Wade Harper) and subcloned into pGEX-4 T-2 vector (Clontech). USP13 site mutants were generated by site-directed mutagenesis (Stratagene).
The anti-USP13 (GTX118595, dilution: 1:500) and anti-Rad51 (N1C2, dilution: 1:200) antibodies were purchased from Genetex. Anti-Ub (P4D1, dilution: 1:500), anti-RPA32 (9H8, dilution: 1:200) and anti-BRCA1 (D9, dilution: 1:200) antibodies were purchased from Santa Cruz Biotechnology. Anti-γH2AX (05-636, dilution: 1:500), anti-FK2 (04-263, dilution: 1:500) and anti-MDC1 (05-1572, dilution: 1:200) were purchased from Millipore. Anti-RAP80 (A303-763A, dilution: 1:500) and anti-53BP1 (A300-272A, dilution: 1:500) were purchased from Bethyl Laboratories. Anti-FLAG (F1804, dilution: 1:1,000), anti-HA (H9658, dilution: 1:1,000), and anti-β-actin (A1978, dilution: 1:2,000) antibodies were purchased from Sigma. Anti-RNF8 (ab4183, dilution: 1:500) was purchased from Abcam.

Li Y., Luo K., Yin Y., Wu C., Deng M., Li L., Chen Y., Nowsheen S., Lou Z, & Yuan J. (2017). USP13 regulates the RAP80-BRCA1 complex dependent DNA damage response. Nature Communications, 8, 15752.

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Detecting DNA Damage and Apoptosis

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γH2AX and 53BP1 immunostaining was carried out as previously described(Mao et al, 2011 (link)). Anti‐ γH2AX (05–636) and anti‐53BP1 (MAB3804) antibodies were purchased from Millipore. Apoptosis in fibroblasts was measured using the Annexin V Staining Kit (Roche).

Simon M., Yang J., Gigas J., Earley E.J., Hillpot E., Zhang L., Zagorulya M., Tombline G., Gilbert M., Yuen S.L., Pope A., Van Meter M., Emmrich S., Firsanov D., Athreya A., Biashad S.A., Han J., Ryu S., Tare A., Zhu Y., Hudgins A., Atzmon G., Barzilai N., Wolfe A., Moody K., Garcia B.A., Thomas D.D., Robbins P.D., Vijg J., Seluanov A., Suh Y, & Gorbunova V. (2022). A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A. The EMBO Journal, 41(21), e110393.

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Antibody Validation for Cell Biology

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The antibodies used in this study were as follows: anti-Cdh1 (sc-56312, 1:1000 WB, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-γH2AX (05-636, 1:500 IF, 1:1000 IF, Millipore, Billerica, MA, USA); anti-BRCA1 (sc-6954, 1:200 IF, Santa Cruz); anti-Phospho-Chk1(Ser345) (2348S, 1:1000 WB, Cell Signaling, Danvers, MA, USA); anti-cleaved Caspase-3 (9664S, 1:1000 WB, Cell Signaling); anti-cleaved PARP (9664S, 1:1000 WB, Cell Signaling); anti-Actin (66009-1-Ig, 1:5000 WB, Proteintech, Wuhan, China); anti-GAPDH (60004-1-1g, 1:5000 WB, Proteintech, Wuhan, China), and anti-PICH(8886S, 1:200 IF, Cell Signaling); the secondary antibodies conjugated to horseradish peroxidase were used for Western blotting. The secondary antibodies of anti-mouse, or anti-rabbit containg Alexa Fluor 488 or 594 were used for immunofluorescence staining (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Li J., Lan M., Peng J., Xiong Q., Xu Y., Yang Y., Zhou Y., Liu J., Zeng Z., Yang X., Zhang Z., Zhang P., Zhu Q, & Wu W. (2022). Cdh1 Deficiency Sensitizes TNBC Cells to PARP Inhibitors. Genes, 13(5), 803.

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Comprehensive Immunoblotting and Immunofluorescence Protocol

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Immunoblotting: anti-FLAG M2 (F3165) (1/2000), anti-DCNL1 Sigma-Aldrich(Clone 3D7) (1/1000), anti-GAPDH(1/2000), anti-Actin(MAB1501) Millipore (1/1000), anti-Cullin1(718700) (1/1000) and anti-Cullin2(700179) (1/5000) Life Technologies, anti-ACADVL (PA5-29959) ThermoScientific, mouse anti-GFP (ab184519) Abcam (1/2000). Sheep polyclonals were raised against full-length KLHL3, the N-terminus of DCNL3,-4,-5, Cul4A,-B and against the C-terminus of Cul3 and Cul5 and used at 1μg/ml.
Immunofluorescence: mouse anti-FLAG M2 (F3165)(1/1000) Sigma, chicken anti-GFP Abcam(ab13970) (1/1000), sheep anti-DCNL5(1/200), sheep anti-Cullin4A(1/100), anti-γ-H2Ax(05-636) (1/1000) from Millipore, secondary antibodies conjugated to Alexa Fluor 488 or 594 from Life Technologies (1/1000).

Keuss M.J., Thomas Y., Mcarthur R., Wood N.T., Knebel A, & Kurz T. (2016). Characterization of the mammalian family of DCN-type NEDD8 E3 ligases. Journal of Cell Science, 129(7), 1441-1454.

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Antibody Sources for Protein Characterization

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Monoclonal anti-Flag (F3165), anti-HA (H9658) and IgG (M5284) antibodies were purchased from Sigma (MO, USA); anti-Myc (M047–3), anti-histidine (D291–3) and anti-β-actin (PM053) were from MBL (Japan); anti-H2A (39209) was from Active Motif (CA, USA). The polyclonal antibodies anti-p21 (sc-397), anti-USP7 (sc-30164) and anti-USP11 (sc-134928) were from Santa Cruz Biotechnology (TX, USA); anti-γH2AX (05–636) was from Millipore (MA, USA); anti-CHK2 (BS1526), anti-pCHK2 (BS4043) and anti-TFIID (BS2262) were from Bioworld (MN, USA); anti-RING1 (AP14560a) was from Abgent (CA, USA); anti-RAP80 (3746) was from Epitomics (CA, USA); and anti-HSCARG was generated against purified recombinant HSCARG. Protein G was purchased from GE Healthcare (Shanghai, China), the Ni-NTA agarose was from Qiagen (Germany) and the protease inhibitor was from Calbiochem (MA, USA).

Hu B., Li S., Zhang X, & Zheng X. (2014). HSCARG, a novel regulator of H2A ubiquitination by downregulating PRC1 ubiquitin E3 ligase activity, is essential for cell proliferation. Nucleic Acids Research, 42(9), 5582-5593.

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Antibodies and Reagents for EGFR Signaling

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Cisplatin (200 μM) was from Mayne Pharma. Anti-EGFR antibodies used for immunofluorescence were against the extracellular domain antibody purified from the mouse 108 hybridoma (ATCC; 1:200), and rabbit anti-cytoplasmic domain antibodies (4267 Cell Signaling; 1:50). Antibodies used for western blotting were: anti-EGFR #2239 (1:1000), anti-phospho-EGFR-Y1068 #2234 (1:500), anti-phospho-EGFR-T669 #3056 (1:500), anti-p38 #9217 (1:2000), anti-phospho-p38 #4511 (1:1000), anti-Hsp27 #2402 (1:1000), anti-phospho-Hsp27 #9709, and anti-calnexin #2433 (1:1000) all from Cell Signaling. Anti-γH2AX #05-636 (1:100) for immunofluorescence was from Millipore.

Tomas A., Jones S., Vaughan S.O., Hochhauser D, & Futter C.E. (2017). Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation. Journal of Cell Science, 130(15), 2481-2490.

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Chromatin Remodeling Protein Analysis

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An anti-BRG1 antibody (ab70558) was purchased from Abcam (San Francisco, CA). Anti-γH2AX (05-636) and anti-H2A (07-146) antibodies were obtained from Merck Millipore (CA). An anti-actin antibody (A5441) and MNase were purchased from Sigma Aldrich (St Louis, MO). Puromycin, etoposide (ETO), and 4-hydroxy tamoxifen (4OHT) were obtained from Sigma Aldrich (St Louis, MO).

Qi W., Chen H., Lu C., Bu Q., Wang X, & Han L. (2018). BRG1 Promotes chromatin remodeling around DNA damage sites. Animal Cells and Systems, 22(6), 360-367.

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Immunohistochemical Analysis of Tumor Samples

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Paraffin-embedded tumor tissues were cut into 5-μM-thick sections, and IHC staining was performed using RTU Vectastain Kit (Vector Laboratories, Newark, CA, USA) according to the manufacturer’s instructions. Briefly, the sections were deparaffined in xylene and rehydrated in graded ethanol. After inactivation of endogenous peroxidase by H2O2 and antigen retrieval, slides were blocked in 5% goat serum and incubated with primary antibody including anti-Rictor (A300-459A, Bethyl Laboratories, 1:500), anti-Ki67(#9449, Cell signaling technology, 1:1000) and anti-γH2AX(05-636, EMD Millipore, 1:600) at 4°C overnight. Then, the slides were incubated with pan-anti-mouse/rabbit/goat secondary antibody (#BA-1300, included in the RTU Vectastain Kit) and visualized using 3,3-diaminobenzidine tetrahydrochloride (DAB) staining solution.

BU C., ZHAO L., WANG L., YU Z, & ZHOU J. (2023). mTORC2 promotes pancreatic cancer progression and parp inhibitor resistance. Oncology Research, 31(4), 495-503.

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Quantification of DNA Damage Foci

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Cells of 1.0 × 10⁵ were plated onto a glass coverslip placed into the well of a 6-well plate. The cells on coverslips were fixed, permeabilized, blocked and washed with phosphate-buffered saline (PBS). Anti-γH2AX (05-636, Merck Millipore, Darmstadt, Germany) was used as the primary antibody and an Alexa Fluor^® 488 secondary antibody (Life Technologies, MA, USA) was used for incubation in the dark. The nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) prior to the examination and image acquisition under a confocal system (Leica Microsystems TCS SP8. Wetzlar, Germany). Control samples without adding the primary antibody were prepared for determining the level of non-specific noise.

Chang S., Huang J., Niu H., Wang J., Si Y., Bai Z., Cheng S, & Ding W. (2020). Epigenetic regulation of osteopontin splicing isoform c defines its role as a microenvironmental factor to promote the survival of colon cancer cells from 5-FU treatment. Cancer Cell International, 20, 452.

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Immunofluorescence Staining of Isolated HSCs

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Isolated HSCs were spread on glass slides using a CytoSpin4 centrifuge (Thermo Scientific, Waltham, MA). For immunofluorescence staining, cells were grown on poly-D-lysine–coated culture slides (BD Pharmingen, San Diego, CA), washed in PBS, fixed in PBS containing 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked in PBS containing 5% bovine serum albumin. Cells were incubated with indicated primary antibodies for 2 hours at room temperature, washed with PBS, and incubated with Alexa-568 and Alexa-488-conjugated secondary antibodies for 1 hour (Life Technologies, Carlsbad, CA). Cells were then washed with PBS and mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA). Images were acquired from a Zeiss AxioImager M2 microscope system equipped with a Plan-Apochromat 63×/NA 1.40 objective, an AxioCamMRm CCD camera, and AxioVision software (Carl Zeiss, Oberkochen, Germany). Anti-γH2AX (05-636, EMD Millipore, Billerica, MA) and anti-phospho-histone H3 (06-570, EMD Millipore, Billerica, MA) antibodies were purchased from the indicated vendors. The anti-TRF1 was a kind gift from Dr. Titia de Lange. Results presented in figures are representative examples for at least two replicated experiments using different cell lines or mice. The exact n number is documented in figure legends.

Zhang S., Matsunaga S., Lin Y.F., Sishc B., Shang Z., Sui J., Shih H.Y., Zhao Y., Foreman O., Story M.D., Chen D.J, & Chen B.P. (2015). Spontaneous Tumor Development in Bone Marrow Rescued DNA-PKcs3A/3A Mice Due to Dysfunction of Telomere Leading Strand Deprotection. Oncogene, 35(30), 3909-3918.

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