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Anti phospho γ h2ax ser139

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Anti-phospho-γ-H2AX (Ser139) is a lab equipment product that detects phosphorylated H2AX at serine 139, a marker of DNA double-strand breaks. It can be used for various research applications.

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Lab products found in correlation

Luminata forte, by Merck Group (1 mentions) Anti vinculin, by Merck Group (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Anti β actin, by Abcam (1 mentions) Anti pchk1 ser345, by Cell Signaling Technology (1 mentions) Anti pum2, by Fortis Life Sciences (1 mentions) Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions) Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Anti 53bp1, by Novus Biologicals (1 mentions) Lsm 510 confocal microscope, by Zeiss (1 mentions) Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions) Vectashield hardset antifade mounting medium, by Vector Laboratories (1 mentions) Ribociclib, by MedChemExpress (1 mentions) Niraparib, by MedChemExpress (1 mentions) H2dcfda, by MedChemExpress (1 mentions) Bromodeoxyuridine (brdu), by MedChemExpress (1 mentions) Palbociclib, by MedChemExpress (1 mentions) Anti histone h3 ab1791, by Abcam (1 mentions)

Spelling variants of this product

Anti-phospho-H2AX (Ser139) (11 citations) Anti-H2AX (11 citations) Phospho-H2AX (ser139) (9 citations) Anti-phospho-H2AX (9 citations) Phospho-H2AX (4 citations) Anti-phospho-H2AX (γ-H2AX; (2 citations) Anti-phospho-γ-H2AX (Ser139) antibody (2 citations)

5 protocols using anti phospho γ h2ax ser139

1

Western Blot Analysis of DNA Damage Response

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Whole cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were resolved on 10–15% SDS-PAGE gels and electroblotted onto PVDF membrane. Membranes were blocked with 5% BSA or dried skimmed milk in TBS-T and incubated in primary antibodies in blocking buffer: anti-pRPA32-Ser33 (A300-246A Bethyl Laboratories), anti-phospho-γH2AX-Ser139 (Merck Millipore, 05–636), anti-pCHK1-Ser345 (Cell signalling #2348), anti-RNA polymerase II CTD repeat YSPTSPS (phospho-S5) (Abcam ab5401), anti-PUM2 (Bethyl Laboratories A300-202A), anti-RNASEH1 (SantaCruz Biotechnologies, H-4, sc-376,326), anti-β-actin (Abcam ab6276), anti-Vinculin (Merck Millipore, V9131), anti-GAPDH (Cell Signalling 14C10 #2118). Membranes were washed and incubated with HRP-conjugated secondary antibodies and blots developed with Luminata™ forte (Merck-Millipore) and imaged using iBright (Invitrogen).
Fletcher C.E., Deng L., Orafidiya F., Yuan W., Lorentzen M.P., Cyran O.W., Varela-Carver A., Constantin T.A., Leach D.A., Dobbs F.M., Figueiredo I., Gurel B., Parkes E., Bogdan D., Pereira R.R., Zhao S.G., Neeb A., Issa F., Hester J., Kudo H., Liu Y., Philippou Y., Bristow R., Knudsen K., Bryant R.J., Feng F.Y., Reed S.H., Mills I.G., de Bono J, & Bevan C.L. (2022). A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer. Molecular Cancer, 21, 82.
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2

Visualizing DNA damage response foci

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Cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, blocked with 2% BSA and 10% goat serum in TBS and incubated with primary antibodies in blocking buffer: anti-phospho-γH2AX-Ser139 (Merck Millipore, 05–636), anti-53BP1 (Novus Biologicals, NB100–904), anti-DNA:RNA hybrid [S9.6] (ENH002, Kerafast). Slides were TBS-washed, incubated in secondary antibodies in blocking buffer (anti-mouse-AlexaFluor-488, anti-mouse-AlexaFluor-594, anti-rabbit-AlexaFluor-488, anti-rabbit-AlexaFluor-594, ThermoFisher) and counterstained with TO-PRO-3 (ThermoFisher, T3605) and DAPI (ThermoFisher, 62,248). Slides were mounted in Vectashield Hardset Antifade Mounting medium (Vector laboratories). Images were acquired using LSM510 confocal microscope (Zeiss) at × 60 magnification. DNA damage foci were quantified using ImageJ.
Fletcher C.E., Deng L., Orafidiya F., Yuan W., Lorentzen M.P., Cyran O.W., Varela-Carver A., Constantin T.A., Leach D.A., Dobbs F.M., Figueiredo I., Gurel B., Parkes E., Bogdan D., Pereira R.R., Zhao S.G., Neeb A., Issa F., Hester J., Kudo H., Liu Y., Philippou Y., Bristow R., Knudsen K., Bryant R.J., Feng F.Y., Reed S.H., Mills I.G., de Bono J, & Bevan C.L. (2022). A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer. Molecular Cancer, 21, 82.
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3

DNA Damage and Cell Cycle Assay

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H2DCFDA (Cat. number: HY-D0940), BrdU (Cat. number: HY-15910), palbociclib (Cat. number: HY-50767), ribociclib (Cat. number: HY-15777), niraparib (Cat. number: HY-10619), and olaparib (Cat. number: HY-10162) were from MedChem Express. Anti-phospho-γ-H2AX (Ser139) (Cat. number: 05-636) and FITC-conjugated anti-BrdU (Cat. number: MAB3262F) antibodies were from Millipore. Anti-Rb (ab181616), Anti-phospho-Rb (Ser780) (ab173289), Anti-histone H3 (ab1791), and Anti- PARP1 (ab191217) were from Abcam.
Li S., Zhang Y., Wang N., Guo R., Liu Q., Lv C., Wang J., Wang L, & Yang Q.K. (2020). Pan-cancer analysis reveals synergistic effects of CDK4/6i and PARPi combination treatment in RB-proficient and RB-deficient breast cancer cells. Cell Death & Disease, 11(4), 219.
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4

Immunoblotting for DNA Repair Proteins

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Olaparib (AZD2281), rucaparib, and pinometostat (EPZ5676) were obtained from Selleckchem. The following antibodies and reagents were obtained from the indicated suppliers: anti-BRCA2 (Bethyl Laboratories, cat. No. A311-267A, 1:5000), anti-BRCA2 ab-1 (Millipore, cat. No. OP95, 1:1000), anti-BRCA2 ab-2 (Millipore, cat. No. CA1033), anti-β-actin (Sigma, cat. No. A1978), anti-GAPDH (Millipore, cat. No. MAB374, 1:20000), anti-DOT1L (Cell Signaling, cat. No.77087), anti-RAD51 (Millipore, cat. No. ABE257 1:500), and anti-BRCA1 (Calbiochem, cat. No. OP92, 1:500), anti-phospho-γH2AX (Ser139) (Millipore, cat. No. 05-636, 1:400), anti-Vinculin (Cell signaling, cat. No. 13901, 1:1000), anti-H3K79Me (Abcam, cat. No. Ab177185 1:1000) and anti-BrdU (BD Biosciences, cat. No. 347583).
Park P.H., Yamamoto T.M., Li H., Alcivar A.L., Xia B., Wang Y., Bernhardy A.J., Turner K.M., Kossenkov A.V., Watson Z.L., Behbakht K., Casadei S., Swisher E.M., Mischel P.S., Johnson N, & Bitler B.G. (2019). Amplification of the mutation-carrying BRCA2 allele promotes RAD51 loading and PARP inhibitor resistance in the absence of reversion mutations.. Molecular cancer therapeutics, 19(2), 602-613.
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5

Plasmid Preparation and Gene Knockdown

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The DAB2IP expression plasmid was prepared as described previously (21 (link)), and expression plasmid for human PARP-1 was obtained from Dr. Binhua Zhou (University of Kentucky College of Medicine, KY, USA) (22 (link)). Gene knockdown was performed using pLKO.1 plasmid expressing gene-specific shRNA and purchased from the National RNAi Core Facility of Taiwan (Academia Sinica, Taipei, Taiwan). Cells were plated with 70% confluence and transfection was carried out by using Xfect (Clontech) according to the manufacturer’s instructions.
Primary antibodies used were as follows: rabbit polyclonal anti-RAD51, mouse polyclonal anti-PARP-1 and monoclonal anti-BRCA1 (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-ubiquitine (Cell signaling), anti-phospho-γ-H2AX (Ser139) (Millipore), mouse monoclonal anti-Flag and anti-actin (Sigma-Aldrich), and mouse monoclonal anti-Ku70 and 80 were home-made antibodies.
Yun E.J., Lin C.J., Dang A., Hernandez E., Guo J., Chen W.M., Allison J., Kim N., Kapur P., Brugarolas J., Wu K., He D., Lai C.H., Lin H., Saha D., Baek S.T., Chen B.P, & Hsieh J.T. (2019). Down-regulation of human DAB2IP Gene Expression in Renal Cell Carcinoma Results in Resistance to Ionizing Radiation. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(14), 4542-4551.
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