Clone:
REA502
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
ICFC, MICS
Alternative names:
H2AFX, H2AX, H2a/x, H2A.X, γ-H2AX, γH2AX

Extended validation for H2AX pS139 Antibody, anti-human/mouse, REAfinity™

Specificity

Epitope competition
In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.
Other clonesOverlap in epitope recognition with REA502
2F3++
N1-431+
2207D (rabb IgG)-
Cells were incubated with an excess of purified unconjugated H2AX pS139 (REA502) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison
Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.
View details
Flow cytometric comparison of different clones for H2AX pS139. HeLa cells were first stained with Viobility™ Fixable Dye followed by fixation and permeabilization using the Cell Signaling Buffer Set A. Cells were then stained with H2AX pS139 antibodies and plotted against the side scatter. As a control,H2AX pS139 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for H2AX pS139. HeLa cells were first stained with Viobility™ Fixable Dye followed by fixation and permeabilization using the Cell Signaling Buffer Set A. Cells were then stained with H2AX pS139 antibodies and plotted against the side scatter. As a control,H2AX pS139 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for H2AX pS139. HeLa cells were first stained with Viobility™ Fixable Dye followed by fixation and permeabilization using the Cell Signaling Buffer Set A. Cells were then stained with H2AX pS139 antibodies and plotted against the side scatter. As a control,H2AX pS139 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for H2AX pS139. HeLa cells were first stained with Viobility™ Fixable Dye followed by fixation and permeabilization using the Cell Signaling Buffer Set A. Cells were then stained with H2AX pS139 antibodies and plotted against the side scatter. As a control,H2AX pS139 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
Flow cytometric comparison of different clones for H2AX pS139. HeLa cells were first stained with Viobility™ Fixable Dye followed by fixation and permeabilization using the Cell Signaling Buffer Set A. Cells were then stained with H2AX pS139 antibodies and plotted against the side scatter. As a control,H2AX pS139 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Specifications for H2AX pS139 Antibody, anti-human/mouse, REAfinity™

Overview

Clone REA502 recognizes the human and mouse histone H2AX antigen phosphorylated at serine 139 (pS139). H2AX is a 14 kDa basal histone and a variant of the H2 histone family. It replaces conventional H2A in a subset of nucleosomes. When cells are exposed to ionizing radiation or DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of H2AX at serine 139 (γH2AX). Because phosphorylation of H2AX is abundant, fast, and correlates well with each DSB, it is the most sensitive marker that can be used to examine the DNA damage produced and the subsequent repair of the DNA lesion. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression.
Additional information: Clone REA502 displays negligible binding to Fc receptors.

Alternative names

H2AFX, H2AX, H2a/x, H2A.X, γ-H2AX, γH2AX

Detailed product information

Technical specifications

CloneREA502
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (I), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, mouse
AntigenH2AX pS139
Alternative names of antigenH2AFX, H2AX, H2a/x, H2A.X, γ-H2AX, γH2AX
Molecular mass of antigen [kDa]15(same molecular weight for human and mouse/rat)
Entrez Gene ID3014, 15270
RRIDAB_2733666, AB_2876938, AB_2876939, AB_2733665

References for H2AX pS139 Antibody, anti-human/mouse, REAfinity™

Publications

  1. Paull, T. T. et al. (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10(15): 886-895
  2. Mannironi, C. et al. (1989) H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3' processing signals. Nucleic Acids Res. 17(22): 9113-9126
  3. Sharma, A. et al. (2012) Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol. Biol. 920: 613-626

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