Non-treated Jurkat cells, and staurosporine treated Jurkat cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:10000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as loading control.
Non-treated Jurkat cells, and staurosporine treated Jurkat cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:10000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as loading control.
WB analysis of C6 using 83307-2-RR
C6 cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours.
C6 cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours.
IHC staining of mouse spleen using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded mouse spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
Immunohistochemical analysis of paraffin-embedded mouse spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IHC staining of rat spleen using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded rat spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
Immunohistochemical analysis of paraffin-embedded rat spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IHC staining of Jurkat using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded Jurkat cells slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:4000 (under 10x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
Immunofluorescent analysis of (4% PFA) fixed UV treated HeLa cells using Phospho-Histone H2A.X (Ser139) antibody (83307-2-RR, Clone: 5N19 ) at dilution of 1:400 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2), CL594-Phalloidin (red).
FC experiment of Jurkat using 83307-2-RR
1x10^6 Jurkat cells untreated (dashed lines) or treated with Staurosporine which intracellularly stained with 0.06 ug Phospho-Histone H2A.X (Ser139) Recombinant antibody (83307-2-RR, Clone:5N19) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.06 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
1x10^6 Jurkat cells untreated (dashed lines) or treated with Staurosporine which intracellularly stained with 0.06 ug Phospho-Histone H2A.X (Ser139) Recombinant antibody (83307-2-RR, Clone:5N19) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.06 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
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Jurkat cells, mouse spleen tissue, rat spleen tissue Note: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0
Positive IF/ICC detected in
UV treated HeLa cells
Positive FC (Intra) detected in
Staurosporine treated Jurkat cells
Recommended dilution
Application
Dilution
Western Blot (WB)
WB : 1:5000-1:50000
Immunohistochemistry (IHC)
IHC : 1:2000-1:8000
Immunofluorescence (IF)/ICC
IF/ICC : 1:200-1:800
Flow Cytometry (FC) (INTRA)
FC (INTRA) : 0.06 ug per 10^6 cells in a 100 µl suspension
It is recommended that this reagent should be titrated in each testing system to obtain optimal results.
Sample-dependent, Check data in validation data gallery.
83307-2-RR targets Phospho-Histone H2A.X (Ser139) in WB, IHC, IF/ICC, FC (Intra), ELISA applications and shows reactivity with human, mouse, rat samples.
PBS with 0.02% sodium azide and 50% glycerol, pH 7.3.
Storage Conditions
Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20oC storage. 20ul sizes contain 0.1% BSA.
Background Information
The histone variant H2AX is a major component of the DNA damage response (DDR), especially functioning in amplifying DNA damage signals. In response to DNA double-strand breaks (DSBs), H2AX is instantaneously phosphorylated at Ser139 (a form called cH2AX) by the kinases ATM and ATR. The phosphorylation of H2AX at Ser139, resulting in the formation of gamma-H2AX puncta in the nuclei, is an early event in the cellular response to DNA damage. Therefore, phospho-Histone H2A. X (Ser139) is also known as γH2AX. The phosphorylation site of H2AX, Ser139, has also been described as Ser140 in other literature, and they recognize the same amino acid site. (PMID: 22908299, PMID: 30106130, PMID:22941631)
Protocols
Product Specific Protocols
WB protocol for Phospho-Histone H2A.X (Ser139) antibody 83307-2-RR
Non-treated Jurkat cells, and staurosporine treated Jurkat cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:10000 incubated at room temperature for 1.5 hours. The membrane was stripped and re-blotted with Alpha Tubulin (66031-1-Ig) antibody as loading control.
WB analysis of C6 using 83307-2-RR
C6 cells were subjected to SDS PAGE followed by western blot with 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:2000 incubated at room temperature for 1.5 hours.
IHC Figures
IHC staining of mouse spleen using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded mouse spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IHC staining of rat spleen using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded rat spleen tissue slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:500 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IHC staining of Jurkat using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded Jurkat cells slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:4000 (under 10x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IHC staining of Jurkat using 83307-2-RR
Immunohistochemical analysis of paraffin-embedded Jurkat cells slide using 83307-2-RR (Phospho-Histone H2A.X (Ser139) antibody) at dilution of 1:4000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).
IF/ICC Figures
IF Staining of HeLa using 83307-2-RR
Immunofluorescent analysis of (4% PFA) fixed UV treated HeLa cells using Phospho-Histone H2A.X (Ser139) antibody (83307-2-RR, Clone: 5N19 ) at dilution of 1:400 and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2), CL594-Phalloidin (red).
FC (INTRA) Figures
FC experiment of Jurkat using 83307-2-RR
1x10^6 Jurkat cells untreated (dashed lines) or treated with Staurosporine which intracellularly stained with 0.06 ug Phospho-Histone H2A.X (Ser139) Recombinant antibody (83307-2-RR, Clone:5N19) and CoraLite®488-Conjugated Goat Anti-Rabbit IgG(H+L) (SA00013-2)(red), or 0.06 ug Rabbit IgG Isotype Control Recombinant Antibody (98136-1-RR, Clone: 240953C9) (blue). Cells were fixed with 4% PFA and permeabilized with 90% MeOH.
The species listed in Tested Reactivity are in-house verified and applicable species. For unlisted species, please refer to the homology analysis of the immunogen sequence and related species. For rabbit polyclonal antibodies, homology >70% is recommended. For mouse monoclonal antibodies and rabbit recombinant antibodies, homology >90% is recommended. Generally, the higher the homology, the greater the applicability. However, there will be certain differences in protein expression in different species, tissues or cells. Therefore, the homology analysis results are for reference only and do not serve as a guarantee.
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