Anti JMJD6 (Santa Cruz, sc28349), anti UBF (Bethyl, A301-859A), anti histone H3 (Abcam, Ab1791), anti BrdU (Sigma, B2531), anti gamma H2AX (Cell signaling, 9718(20E3)), anti GAPDH (Chemicon, MAb374), anti myc (Santa Cruz, sc-40), anti Treacle (Santa Cruz, sc374536), anti V5 (Cell Signaling, 12032), anti NBS1 (Sigma, PLA0179), anti phospho ATM (S1981) (Cell signaling, 10H11.E12).Anti Rad51 (Millipore, PC130), Anti RPA S33 (Bethyl, A306-246A-T)
Anti gamma h2ax
Manufactured by Cell Signaling Technology
Sourced in Germany, United Kingdom
Anti-gamma-H2AX is a laboratory reagent that detects the phosphorylated form of the histone H2AX, known as gamma-H2AX. Gamma-H2AX is a marker of DNA double-strand breaks and is used to study the cellular response to DNA damage.
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Lab products found in correlation
Anti p21, by Abcam (2 mentions)
Anti phospho atm, by Cell Signaling Technology (1 mentions)
Anti brdu, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Anti v5, by Cell Signaling Technology (1 mentions)
Anti jmjd6, by Santa Cruz Biotechnology (1 mentions)
Anti nbs1, by Merck Group (1 mentions)
Anti rad51, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti trf1, by Santa Cruz Biotechnology (1 mentions)
Anti blm, by Santa Cruz Biotechnology (1 mentions)
Anti rpa70, by Cell Signaling Technology (1 mentions)
Anti rad51, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti pml, by Santa Cruz Biotechnology (1 mentions)
Ecl staining, by Bio-Rad (1 mentions)
Ripa buffer, by Roche (1 mentions)
Anti areg, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
7 protocols using anti gamma h2ax
1
Comprehensive Antibody Staining Protocol
2
Colibactin Induces Cellular Senescence
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The cellular senescence induced by colibactin was with the associated cell enlargement called megalocytosis and was determined for every EcN mutant constructed in this study in the Mcc gene cluster, in the iroA locus, and for the clbP-S95R mutant. As previously described [67 (link)], HeLa cells (ATCC, CCL-2) were infected for 4 hours. The cells were then washed and incubated with gentamicin for 72 hours before staining with Giemsa. The genotoxicity of EcN and the clbP-S95R chromosomal mutant was confirmed by an In-Cell Western procedure, as previously described [38 ]. In brief, HeLa cells were infected in 96-well plates for 4 hours at a given multiplicity of infection (number of bacteria per cell at the onset of infection). Four hours after the end of infection cells were fixed, permeabilized and stained with rabbit monoclonal anti-gamma-H2AX (Cell Signaling, 20E4, 1:200) followed by an infrared fluorescent secondary antibody. DNA was counterstained with RedDot2 (Biotum). Fluorescence was recorded with an Odyssey infrared imaging system (Li-Cor).
3
Immunofluorescence Assay of DNA Repair Factors
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Cells were fixed 24 hours after transfection. Fixed cells were processed for immunofluorescence. The following antibodies were used: anti-FLAG (Sigma, F7425 or F1804), anti-RPA70 (Cell Signaling, 2267), anti-RPA32 pS33 (Abcam, ab87278), anti-BLM (Santa Cruz, SC-7790), anti-Rad51 (Santa Cruz SC-8349), anti-RecQL4 (Abcam, ab34800), anti-gamma H2AX (Cell Signaling, 2577), and anti-TRF1 (Santa Cruz, SC-6165). Primary antibodies were diluted 1:1000, except the in-house ATRIP antibody, which was diluted 1:20. Secondary antibodies (Alexa Fluor, Invitrogen) were diluted 1:2000.
4
Western Blot Analysis of Cellular Proteins
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As described previously [20 (link)], cell pellets were lysed in 100 µl RIPA buffer (Roche, Mannheim, Germany) for 15 min at 4 °C. Cell fragments were removed by centrifugation (13,000× g rpm, 10 min, 4 °C), and the supernatants were collected. Then, 40 µg of total RIPA lysates were loaded on polyacrylamide gels. After separation, the gel was blotted onto a PVDF membrane. Each blot was blocked for 1 h with 5% milk powder/TBS-T and incubated overnight at 4 °C with anti-AREG (1:500, Santa Cruz Biotech, Heidelberg, Germany), anti-PML (1:200, Santa Cruz Biotech, Heidelberg, Germany), anti-H3K9 (1:500, Merck KGaA, Darmstadt, Germany), anti-gamma-H2AX (1:1000, Cell Signaling Technology, Frankfurt, Germany), anti-p21 (1:1000, Abcam, Berlin, Germany), or anti-β-actin (1:5000, Sigma-Aldrich) in 3% MP/TBST. After washing three times with TBST, the membrane was incubated with a horseradish peroxidase-coupled secondary antibody (1:2000, anti-rabbit HRP or anti-mouse HRP, Cell Signaling Technology) for 1 h. The immunoreactions were visualized by ECL staining (Bio-Rad, Feldkirchen, Germany).
For competitive Western blot, 400 ng of AREG antibody was pre-incubated with 4 µg of recombinant AREG (Sigma Aldrich, Germany) for 3 h at 4 °C before overnight incubation with the PVDF membrane.
For competitive Western blot, 400 ng of AREG antibody was pre-incubated with 4 µg of recombinant AREG (Sigma Aldrich, Germany) for 3 h at 4 °C before overnight incubation with the PVDF membrane.
5
Comprehensive Immunostaining Protocol
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Primary antibodies were γ-H2AX (Abcam, Cambridge, UK, Anti-gamma H2A.X (S139 antibody [9F3], ab26350) or GAPDH (14C10) rabbit IgG mAb (Cell Signaling Technology, Cambridge, UK CST, 2118), anti-ckit (Abcam, ab32363), Anti-eNOS (Cell Signaling Technology, 9572), or PDGFR-b (Abcam, ab69506); anti-beta Actin (Abcam, ab8227), anti-IL6 (Abcam, ab6672), anti-phospho-Rb (Abcam, ab47763), anti-p21 (Abcam, ab109520), or anti-CD163 (Abcam, ab156769); anti-mTOR (Abcam, ab2732), anti-NFkB (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc8008), anti-ATM (Abcam, ab32420), or anti-GLUT1 (Abcam, ab115730); CD34 (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc74499). Secondary antibodies: AF488GAM (Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA; Novus Biogicals, LLC, Centennial, CO, USA, A11029), AF546GAR Molecular Probes, A11035), HRP-anti-mouse IgG (BD PharmigenTM (BD Biosciences), San Jose, CA, USA, 7076S), and HRP-anti-rabbit IgG (BD PharmigenTM, 7074S).
6
Quantifying DNA Damage Response Proteins
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AGO1552s were trypsinised off Mylar, lysed and protein concentration determined using the Bradford assay. Lysates were then run on 12% acrylamide gels (8% for p-ATM), transferred to a nitrocellulose membrane, blocked in 5% milk followed by incubation overnight at 4 0 C with antigamma-H2AX (see above), anti-p-ATM (see above), anti-Actin, anti-BCL-2, anti-Bax, anti-Caspase 3, anti-Caspase 7, anti-Caspase 9 and anti-p-p53 (Cell Signalling) primary antibodies.
Membranes were then washed in TBS-0.1% tween20 and incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (Cell Signalling). Luminata (Merck Millipore) was used for chemiluminescence and membranes were visualised on a Syngene G:BOX with exposure time set automatically to prevent saturation. AGO1552 cells were used for western blotting to as the number of alpha particles delivered to each adhering fibroblast cell could be kept far more consistent than with lymphocytes in suspension.
Membranes were then washed in TBS-0.1% tween20 and incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (Cell Signalling). Luminata (Merck Millipore) was used for chemiluminescence and membranes were visualised on a Syngene G:BOX with exposure time set automatically to prevent saturation. AGO1552 cells were used for western blotting to as the number of alpha particles delivered to each adhering fibroblast cell could be kept far more consistent than with lymphocytes in suspension.
7
HALO-POLQ Localization in Irradiated RPE-1 Cells
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HALO-POLQ expressing RPE-1 cells were plated at 70% density on chamber slides 24 hours prior to treatment. Vehicle or olaparib was added immediately prior to irradiation with 10Gy in a RadSource RS2000 irradiator. Cells recovered for two hours post-irradiation prior to harvest. Janelia Fluor 549 HaloTag Ligand (Promega) was added to the recovery media 15 minutes prior to collection. Upon collection, cells were fixed with 4% paraformaldehyde (Electron Microscopy Services) and permeabilized with 0.5% Nonidet P-40 substitute (Fluka). The cells were then blocked with 0.5% BSA (Fisher Bioreagents) and 0.2% fish gelatin (Sigma) prior to primary antibody incubation in blocking solution overnight. Primary antibodies used were anti-CtIP (Novus; NB-79610) and anti-gamma H2AX (Cell Signaling Technology; 9718). Slides were then washed and incubated with Alexa Fluor secondary antibodies in blocking solution. Cells were subsequently washed and stained with DAPI (BioLegend) prior to mounting. Images were acquired on a BX61 Olympus microscopy with recommended Z stack depth optimization. Images were processed with Imaris and FIJI software packages.
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