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Anti phospho h2ax s139

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Anti-phospho-H2AX (S139) is a laboratory product that detects phosphorylation of the histone variant H2AX at serine 139. This phosphorylation is a well-established marker of DNA double-strand breaks.

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Lab products found in correlation

Lumiglo chemiluminescent substrate, by Cell Signaling Technology (3 mentions) Anti ku80, by Thermo Fisher Scientific (3 mentions) Anti ku70, by Thermo Fisher Scientific (3 mentions) Anti phospho chk2 t68, by Cell Signaling Technology (2 mentions) Anti tubulin, by Merck Group (2 mentions) Hrp linked anti mouse igg, by Cell Signaling Technology (2 mentions) Anti kap1, by Fortis Life Sciences (2 mentions) Anti kap1 phospho s824, by Fortis Life Sciences (2 mentions) Anti phospho atm s1981, by Abcam (2 mentions) Anti histone h3 antibody, by BioLegend (2 mentions) Anti dna pkcs, by Thermo Fisher Scientific (2 mentions) Horseradish peroxidase hrp, by GE Healthcare (2 mentions) Anti lamin b, by Abcam (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti sapk jnk, by Cell Signaling Technology (1 mentions) Anti p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions) Anti brdu fitc, by BioLegend (1 mentions) P38 mapk, by Cell Signaling Technology (1 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Anti chk1 sc 8408, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

Anti-H2AX (11 citations) Anti-phospho-H2AX (9 citations) Phospho-H2AX (4 citations) Mouse monoclonal anti-phospho-S139-H2AX antibody (3 citations) Phospho-S139-H2AX (2 citations)

8 protocols using anti phospho h2ax s139

1

Antibody Characterization for Cell Signaling Assays

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Antibodies used in this study are as follows. Anti-human Claspin was generated against the human recombinant Claspin with aa896–1,014 produced in E. coli. Anti-Chk1 phospho-S345 (#2348), anti-Chk1 phospho-S317 (#2344), anti-p44/42 MAPK (Erk1/2) (#4695), anti- SAPK/JNK (#9252), p38 MAPK (#8690), anti-p38 MAPK T180/Y182 (#4511), anti-p44/42 MAPK (Erk1/2) T202/Y204 (#4370), anti-SAPK/JNK T183/Y185 (#4668), Caspase-9 (#9508), Cleaved Caspase-3(#9661), and Mcl-1 (#5453) were obtained from Cell Signaling. Anti-α Tubulin (sc23948), anti-MCM2 (sc-9839), and anti-Chk1 (sc-8408) were obtained from Santa Cruz. Anti-phospho-H2A.X S139 (06-536) was purchased from Merck. Anti-BrdU (Ab6326) was purchased from Abcam. Anti-ATR phospho-T1989 (GTX128145) was purchased from GeneTex. Anti-BrdU (555627) was purchased from BD Pharmingen. Anti-H2A.X phospho-S139 (613402), anti-Rat IgG Alexa Fluor 555 (405420), and FITC-Anti-BrdU (364104) were purchased from Biolegend. RPA32 phospho-S4/S8 (A300-245A) and anti-MCM2 S53(A300-756A) were purchased from Bethyl. Anti-Mouse IgG Alexa Fluor 488 (A-11017) was purchased from Invitrogen. Goat Anti-Rabbit IgG HRP (111-035-003) and Goat Anti-Mouse IgG (115-035-003) were purchased from Jackson ImmunoResearch Laboratory.
Hsiao H.W., Yang C.C, & Masai H. (2023). Claspin-Dependent and -Independent Chk1 Activation by a Panel of Biological Stresses. Biomolecules, 13(1), 125.
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2

DNA Damage Response Protein Analysis

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The following commercial antibodies were used: anti-DNA-PKcs phospho-S2056 (Abcam, ab124918), anti-MRE11 (Abcam, ab214), anti-RAD50 (BD Bioscience, 611010), anti-NBS1 (BD Bioscience, 611871), anti-ATM (Abcam, ab109027), anti-ATM phospho-S1981 (Abcam, ab81292), anti-MDC1 (Abcam, ab5003), anti-KAP1 (Bethyl Laboratories, A300–274A), anti-KAP1 phospho-S824 (Bethyl Laboratories, A300–767A), anti-phospho-H2AX (S139) (EMD Millipore, 05–636), anti-γH2AX (Cell Signaling Technology, 7631), anti-tubulin (Sigma, T5168), anti-CHK2 phospho-T68 (Cell Signaling Technology, 2197), anti-CHK2 antibody (Cell Signaling Technology, 2662), anti-XLF (Cell Signaling Technology, 2854), anti-LIG4 antibody (Cell Signaling Technology, 14649), anti-Histone H3 antibody (Cell Signaling Technology, 9715), anti-XRCC4 antibody (Santa Cruz, sc-271087), and anti-EXO1 (Thermo Fisher Scientific, MA5–12262). In-house produced antibodies include mouse monoclonal antibodies against DNA-PKcs (25–4), Ku80, and Ku70. Secondary antibodies included anti-mouse IgG (HRP-linked) and anti-rabbit IgG (HRP-linked), which were purchased from Cell Signaling Technology.
Lu H., Saha J., Beckmann P.J., Hendrickson E.A, & Davis A.J. (2019). DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response. Nucleic Acids Research, 47(18), 9467-9479.
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3

Immunoblotting of DNA Repair Proteins

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Immunoblotting was performed as previously described (24 (link)). The following antibodies were used in this study: anti-DNA-PKcs phospho-T4102 (made in this study as described above), anti-DNA-PKcs phospho-S2056 (Abcam, ab124918), anti-ATM phospho-S1981 (Abcam, ab81292), anti-CHK2 phospho-T68 (Cell Signaling, 2197), anti-LIG4 (Cell Signaling, 14649), anti-Ku80 (Santa Cruz, sc-17789), anti-Ku70 (Santa Cruz, sc-515736), anti-XRCC4 (Santa Cruz, sc-271087), anti-XLF (Santa Cruz, sc-166488), anti-GFP (Santa Cruz, sc-8334), anti-KAP1 (Bethyl Laboratories A300-274A), anti-KAP1 phospho-S824 (Bethyl Laboratories, A300-767A), anti-tubulin (Sigma-Aldrich, T5168), anti-FLAG M2 (Sigma-Aldrich, F1804), anti-phospho-H2AX (S139) (EMD Millipore, 05-636), and anti-Histone H3 antibody (Biolegend, 819411). Mouse monoclonal antibodies against DNA-PKcs (Clone # 25-4) were produced in house. Secondary antibodies used include anti-mouse IgG (HRP-linked) (Cell Signaling, 7076) and anti-rabbit IgG (HRP-linked) (Cell Signaling, 7074). Image J (Version 1.53e) was used to quantify the intensities of protein bands in the immunoblots.
Lu H., Zhang Q., Laverty D.J., Puncheon A.C., Augustine M.M., Williams G.J., Nagel Z.D., Chen B.P, & Davis A.J. (2023). ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining. Nucleic Acids Research, 51(13), 6770-6783.
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4

Immunoblotting for DNA Repair Proteins

Cited 1 time
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Immunoblotting was performed as previously described (15 (link)). The following antibodies were used in this study: antibodies from Abcam- anti-DNA-PKcs phospho-S2056 (ab124918), anti-MRE11 (ab214), anti-ATM (ab109027), anti-ATM phospho-S1981 (ab81292); antibodies from Cell Signaling Technology—anti-CHK2 phospho-T68 (2197), anti-LIG4 (14649), anti-Artemis (13381), anti-XLF (2854), anti-phospho S/T-Q motif, anti-mouse IgG (HRP-linked) (7076) and anti-rabbit IgG (HRP-linked) (7074); antibodies from Santa Cruz Biotechnology—anti-XRCC4 (sc-271087), anti-XLF (sc-166488), anti-GFP (sc-8334) and anti-Actin (sc-8432); antibodies from Bethyl Laboratories- anti-KAP1 (A300-274A) and anti-KAP1 phospho-S824 (A300-767A); antibodies from Sigma-Aldrich- anti-tubulin (T5168) and anti-FLAG M2 (F1804); anti-phospho-H2AX (S139) (EMD Millipore, 05-636), and anti-Histone H3 antibody (Biolegend, 819411 or EMD Millipore, 07-690). Anti-RECQL4 was purchased from Genetex (GTX55183). In-house produced antibodies include mouse monoclonal antibodies against DNA-PKcs (Clone #25-4), Ku80, and Ku70 (15 (link)).
Lu H., Guan J., Wang S.Y., Li G.M., Bohr V.A, & Davis A.J. (2022). DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks. Nucleic Acids Research, 50(10), 5635-5651.
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5

Protein Expression Analysis in Cells

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Cells were lysed in mammalian protein extraction reagent (Pierce). After quantification using a BCA protein assay kit (Pierce), total proteins were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore). Membranes were blocked in 5% non-fat milk (Bio-Rad) and then incubated with anti-CDK7 (Cat No: sc-7344, Santa Cruz Biotechnoloy); anti-BRCA1 (Cat No: sc-6954, Santa Cruz); anti-BRCA2 (Cat No: OP95, Millipore); anti-RAD51 (Cat No: ab213, Abcam); anti-Ku80 (Cat No: MA5–12933, Thermo); anti-Ku70 (Cat No: MA5–15110, Thermo); anti-RNAP II (Cat No: sc-17798, Santa Cruz Biotechnoloy); anti-RNAP II p-Ser5 (Cat No: A300–655A-2, Bethyl); or anti-phospho-H2AX(S139) (Cat No:05–636, Clone No: JBW301, Millipore), followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Anti-b-Tubulin (Cat No: 2128, Clone No: 9F3, CST) or anti-Actin (Cat No: A3854, Sigma) was used for internal loading control. Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling).
Shan W., Yuan J., Hu Z., Jiang J., Wang Y., Loo N., Fan L., Tang Z., Zhang T., Xu M., Pan Y., Lu J., Long M., Tanyi J.L., Montone K.T., Fan Y., Hu X., Zhang Y, & Zhang L. (2020). Systematic Characterization of Recurrent Genomic Alterations in Cyclin-Dependent Kinases Reveals Potential Therapeutic Strategies for Cancer Treatment. Cell reports, 32(2), 107884.
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6

Detecting DNA Damage Signaling Proteins

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Western blotting was performed using the following primary antibodies: anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo); anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo); anti-DNA-PKcs (Cat No: MA5-13404, Thermo); anti-phospho-H2AX(S139) (Cat No:05-636, Clone No: JBW301, Millipore); anti-PARP (Cat No:9542, CST); anti-β-Tubulin (Cat No:2128, Clone No:9F3, CST); anti-Lamin B (Cat No:ab8982, Clone No:119D5-F1, abcam) were used, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling). The antibody information was provided in the Supplementary Note 1.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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7

Immunofluorescence Analysis of DNA Damage

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Cells were fixed for 15 min. with 4% paraformaldehyde in PBS and permeabilized with PBST solution (0.5% Triton X‐100 in PBS) for 30 min. After blocking with 5% bovine serum albumin in PBST solution for 1 hr, cells were incubated with the anti‐phospho H2AX (S139) (05‐636; Millipore, Temecula, CA, USA), anti‐53BP1 (sc22760; Santa Cruz Biotechnology), anti‐phospho ATM (S1981) (ab1888; Abcam) antibodies for overnight at 4°C respectively. Antigen was detected with the secondary antibodies conjugated to FITC (F0382; Sigma‐Aldrich) and TRITC (T5393; Sigma‐Aldrich). Cells were coverslipped using VECTASHIELD mounting media plus DAPI (H‐1200; Vector Laboratories, Burlingame, CA, USA). Images were acquired with a confocal microscope (Leica TCS SPE; Leica Microsystems GmbH, Wetzlar, Germany). Percentages of γH2AX foci positive cells were counted in five random high power fields.
Lee K., Jeong J.E., Kim I.H., Kim K, & Ju B. (2015). Cyclo(phenylalanine‐proline) induces DNA damage in mammalian cells via reactive oxygen species. Journal of Cellular and Molecular Medicine, 19(12), 2851-2864.
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8

Detecting DNA Damage Signaling Proteins

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Western blotting was performed using the following primary antibodies: anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo); anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo); anti-DNA-PKcs (Cat No: MA5-13404, Thermo); anti-phospho-H2AX(S139) (Cat No:05-636, Clone No: JBW301, Millipore); anti-PARP (Cat No:9542, CST); anti-β-Tubulin (Cat No:2128, Clone No:9F3, CST); anti-Lamin B (Cat No:ab8982, Clone No:119D5-F1, abcam) were used, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling). The antibody information was provided in the Supplementary Note 1.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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