Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y]

14 product images

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  Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunofluorescence staining of A549 (Human lung carcinoma cell line) labeling gamma H2A.X (phospho S139) (green) with ab81299.
  

  Cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in PBS-0.2% Triton for 10 minutes. After blocked for 1 hour, primary antibody was diluted in blocking buffer (1/100) and incubated with fixed cells overnight at 4°C. Cells were washed and incubated with secondary antibodies (1/100) for 1 hour at room temperature. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was performed using confocal laser scanning microscopy (Lecia) or fluorescence microscopy (Olympus).

  Dexamethasone, DEX; Cisplatin, DDP.

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  Immunoprecipitation - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  ab81299 at 1/40 immunoprecipitating Histone H2A.X (phospho S139) in HepG2 (human hepatocellular carcinoma epithelial) whole cell lysate observed at 15 KDa (lanes 1 and 2).
  

  Lane 1 (input): HepG2 treated with etoposide and TSA whole cell lysate 10μg
  Lane 2 (+): ab81299 + HepG2 treated with etoposide and TSA whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab81299 in HepG2 treated with etoposide and TSA

  For western blotting, ab81299 (Purified) at 1/200 dilution and VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/1000 dilution.

  Blocking buffer and concentration: 5% NFDM/TBST.
  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Predicted band size: 15 kDa

  Exposure time: 30s

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  Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Blocking buffer - 5% NFDM/TBST

  Diluting buffer - 1% BSA

  All lanes: Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/100000 dilution

  Lane 1: HepG2 cell lysate – treated with etoposide at 20 µg

  Lane 2: HepG2 cell lysate – untreated at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 15 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with H₂O₂) labelling Histone H2A.X (phospho S139) with ab81299 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) using Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).
  

  Control 1: primary antibody (1/250) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

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  Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  All lanes: Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/500000 dilution

  Lane 1: Jurkat cell lysate - untreated at 10 µg

  Lane 2: Jurkat cell lysate - treated with etoposide at 10 µg

  Secondary

  All lanes: HRP Labelled goat anti-rabbit at 1/2000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

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  This image is courtesy of a customer review submitted by Carl Hobbs.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  ab81299 staining H2A.X in Human Testis tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in Citric acid. Samples were incubated with primary antibody (1/50 in TBS) for 2 hours at 21°C. A biotin conjugated Anti-Rabbit IgG (goat polyclonal) was used as the secondary antibody at a 1/250 dilution.

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  Dot Blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Dot blot analysis of Histone H2A.X single phospho peptide pS139 (lane 1) and Histone H2A.X non-phospho peptide (lane 2) with ab81299 at 1/1000. Blocking and diluting buffer was 5% NFDM/TBST. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) ab97051 Peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemical staining of paraffin embedded human brain with unpurified ab81299 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemical staining of paraffin embedded human brain with purified ab81299 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using unpurified ab81299 at a dilution of 1/100.
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling gamma H2A.X with ab81299 at 1/3000 (0.352 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human breast carcinoma without lambda protein phosphatase treatment (image A). No signal was detected when tissues were treated with lambda protein phosphatase (image B). The section was incubated with ab81299 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB ab209101. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

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  This image is courtesy of an anonymous customer review

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of 10% neutral buffered formalin fixed mouse testes permeabilzed with Wash buffer, blocking solution and secondary diluent was 1X TBS with 0.1% Tween-20. Stained with ab81299 at 0.125 µg/ml.
  Secondary antibody used was Biotin AffiniPure Donkey Anti-Rabbit IgG (H+L) at 5µg/ml.
  Blocking was done with 10% serum for 15 minutes at 22°C.
  The sample was incubated with the primary antibody for 30 minutes at 22°C with Dako Antibody Diluent (S3022).
  Antigen retrieval method was heat mediated Dako Target Retrieval Solution (S1699)

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling gamma H2A.X with ab81299 at 1/3000 (0.352 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human cervical carcinoma without lambda protein phosphatase treatment (image A). No signal was detected when tissues were treated with lambda protein phosphatase (image B). The section was incubated with ab81299 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB ab209101. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

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  Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

  Mouse anti GAPDH was used as a GAPDH loading control.

  All lanes: Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/1000 dilution

  Lane 1: HepG2 Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

  Lane 2: HepG2 Etoposide treated (100 uM, 90 min) at 20 µg

  Lane 3: Jurkat Vehicle Control Etoposide (0 uM, 90 min) at 20 µg

  Lane 4: Jurkat Treated Etoposide (100 uM, 90 min) at 20 µg

  Lane 5: Human Brain at 20 µg

  Predicted band size: 15 kDa