Phospho-Histone H2A.X (S139) Recombinant Rabbit Monoclonal Antibody [SR33-09]
cat.: ET1602-2
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IHC-P, IF-Cell
Clonality: Monoclonal
Clone number: SR33-09
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 15 kDa
Isotype: IgG
Immunogen: Synthetic phospho-peptide corresponding to residues surrounding Ser139 of Human Histone H2AX.
Positive control: HeLa treated with 20μM Etoposide for 2 hours whole cell lysate, HeLa treated with UV for 2 hours whole cell lysate, NIH/3T3 treated with 25μM Etoposide for 5 hours whole cell lysate, HepG2 cell lysate–treated with etoposide, rat brain tissue, mouse testis tissue, mouse brain tissue, mouse small intestine tissue, human colon carcinoma tissue.
Subcellular location: Nucleus, Chromosome
Recommended Dilutions:
  WB
  IHC-P
  IF-Cell
1:1,000-1:2,000
1:50-1:5,000
1:2,000
Uniprot #: SwissProt: P16104 Human | P27661 Mouse
Unigene: 2850 Rat
Alternative names: AW228881 H2A histone family member X H2A.FX H2A.X H2a/x H2AFX H2AX H2AX histone H2AX_HUMAN Hist5.2ax Histone 2A Histone 2AX Histone H2A.X Histone H2AX RGD1566119 gamma H2A.X γ H2A.X γ-H2A.X
Images
ET1602-2_1.jpg Fig1: Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

Lane 1: HeLa whole cell lysate
Lane 2: HeLa treated with 20μM Etoposide for 2 hours whole cell lysate
Lane 3: HeLa whole cell lysate
Lane 4: HeLa treated with UV for 2 hours whole cell lysate
Lane 5: NIH/3T3 whole cell lysate
Lane 6: NIH/3T3 treated with 25μM Etoposide for 5 hours whole cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15/20 kDa

Exposure time: 53 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1602-2) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
ET1602-2_2.jpg Fig2: Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1602-2, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: HepG2 cell lysate–treated with etoposide
Lane 2: HepG2 cell lysate–untreated
ET1602-2_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/50 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/50 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/400 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_8.jpg Fig8: Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/1,000 dilution.

Lane 1: C6 treated with 25μM Etoposide for 5 hours whole cell lysate
Lane 2: C6 whole cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 20 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1602-2) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1602-2_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded mouse breast tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/5,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1602-2_12.jpg Fig12: Immunocytochemistry analysis of HeLa cells untreated / treated with 20μM etoposide for 2 hours labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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