Cells were fixed with 4% paraformaldehyde solution for 15 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, blocked with 1% BSA for 30 minutes, and then with Anti-γ H2AX (S139) (Millipore, 1:500 diluted in 1% In BSA) for 1 hour at 37 °C. After washing 3 times with PBS, the cells were incubated with the secondary antibody for 1 hour at 37 °C and stained with DAPI to reveal the nucleus. Capture and visualize fluorescence images under a high-content imaging system (Operetta, Perkin Elmer).
Anti γ h2ax s139
Manufactured by Merck Group
Anti-γ H2AX (S139) is a lab equipment product used to detect and quantify the phosphorylation of the histone variant H2AX at serine 139 (γ-H2AX), which is a marker of DNA double-strand breaks. This product can be used in various research applications, including cell biology, molecular biology, and DNA damage response studies.
Automatically generated - may contain errors
Lab products found in correlation
Operetta, by PerkinElmer (1 mentions)
γh2ax s139, by Merck Group (1 mentions)
Hnrnpd, by Merck Group (1 mentions)
Anti 53bp1, by Santa Cruz Biotechnology (1 mentions)
Ccd camera, by Hamamatsu Photonics (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Ab83501, by Abcam (1 mentions)
Anti phospho chk2 t68, by Cell Signaling Technology (1 mentions)
Anti phospho rpa32 s4 s8, by Fortis Life Sciences (1 mentions)
Anti chk2, by Cell Signaling Technology (1 mentions)
Anti h2ax, by Cell Signaling Technology (1 mentions)
Sc 6954, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
4 protocols using anti γ h2ax s139
1
Immunofluorescence Assay for DNA Damage
2
DNA Damage Response Tracking in HeLa Cells
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HeLa cells, grown on glass coverslips, were fixed with 4% paraformaldehyde and permeabilized with 0.5% triton. Samples were blocked 10 min in 1%BSA at RT and incubated 1 h with anti-BrdU (1:200, 347580, BD Biosciences) or anti-γH2Ax S139 (1:600, 05-636, Millipore) 37°C. After washing, samples were incubated 45 min at 37°C with AlexaFluor 594-conjugated chicken anti-rabbit and 488-conjugated rabbit anti-mouse or 555-conjugated goat anti-mouse IgG (H+L) (Life Technologies), and analyzed with a Zeiss LSM100 confocal microscope.
UVC micro-irradiation was performed as described by Suzuki et al. (23 (link)), then the following antibodies were used for protein detection HNRNPD (1:200, 07-260, Millipore), γH2Ax S139 (1:600, 05-636, Millipore).
UVC micro-irradiation was performed as described by Suzuki et al. (23 (link)), then the following antibodies were used for protein detection HNRNPD (1:200, 07-260, Millipore), γH2Ax S139 (1:600, 05-636, Millipore).
3
Immunofluorescent Visualization of DNA Damage Markers
4
Western Blot Analysis of DNA Damage Signaling
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Cell lysates were prepared by sonicating in UTB buffer (8 M urea, 50 mM Tris HCl [pH 7.4], 150 mM β-mercaptoethanol). Following protein concentration determination using the Bradford reagent (Bio-Rad), lysates were suspended in 2× Laemmli sample buffer (Bio-Rad) and heated to 95°C for 10 min. Lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard procedures.
The following primary antibodies were used for Western blotting: anti-K8α/K-bZIP (SAB5300152; Sigma-Aldrich), anti-ORF6/SSB (provided by Gary Hayward), anti-K8.1A (in-house), anti-β-actin (A2228; Sigma-Aldrich), anti-ATM (2873; Cell Signaling), anti-phospho-ATM (S1981) (AF1655; R&D Systems), anti-CHK2 (2662; Cell Signaling), anti-phospho-CHK2 (T68) (2661; Cell Signaling), anti-RPA32 (NA19L; Calbiochem), anti-phospho-RPA32 (S4/S8) (A300-245A; Bethyl), anti-H2AX (7631; Cell Signaling), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 and anti-KU80 (sc-6954; Santa Cruz), and anti-KU70 (ab83501; Abcam). Goat anti-mouse and swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako Laboratories) and ECL detection reagent (GE Healthcare) were used to visualize proteins on a Fusion SL chemiluminescence imaging system (Vilber Lourmat).
The following primary antibodies were used for Western blotting: anti-K8α/K-bZIP (SAB5300152; Sigma-Aldrich), anti-ORF6/SSB (provided by Gary Hayward), anti-K8.1A (in-house), anti-β-actin (A2228; Sigma-Aldrich), anti-ATM (2873; Cell Signaling), anti-phospho-ATM (S1981) (AF1655; R&D Systems), anti-CHK2 (2662; Cell Signaling), anti-phospho-CHK2 (T68) (2661; Cell Signaling), anti-RPA32 (NA19L; Calbiochem), anti-phospho-RPA32 (S4/S8) (A300-245A; Bethyl), anti-H2AX (7631; Cell Signaling), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 and anti-KU80 (sc-6954; Santa Cruz), and anti-KU70 (ab83501; Abcam). Goat anti-mouse and swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako Laboratories) and ECL detection reagent (GE Healthcare) were used to visualize proteins on a Fusion SL chemiluminescence imaging system (Vilber Lourmat).
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