Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22820-23] to Histone H2A.X - ChIP Grade – BSA and Azide free
  • Suitable for: PepArr, ChIP, IHC-P, IP, WB, ICC/IF, Flow Cyt
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free
    See all Histone H2A.X primary antibodies

  • Description

    Rabbit monoclonal [EPR22820-23] to Histone H2A.X - ChIP Grade – BSA and Azide free

  • Host species

    Rabbit

  • Tested Applications & Species

    Application Species
    ChIP
    Human
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    PepArr
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Wild-type HAP1 whole, Histone H2A.X knockout HAP1 whole, Human brain, HeLa, 293T and HEK-293 lysates. IHC-P: Human breast carcinoma and Human testis tissues. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. IP: HeLa cells.

  • General notes

    Ab256544 is the carrier-free version of ab229914. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab256544 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Images

  • Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Western blot - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    All lanes : Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Histone H2A.X knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 15 kDa
    Observed band size: 16 kDa
    why is the actual band size different from the predicted?



    Ab229914 was shown to specifically react with Histone H2A.X in wild-type HAP1 cells as signal was lost in Histone H2A.X knockout cells. Wild-type and Histone H2A.X knockout samples were subjected to SDS-PAGE. ab229914 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.   The blot was developed on a BIO-RAD® ChemiDocâ„¢ MP instrument using the ECL technique. Blocking/Diluting buffer and concentration: 5% NFDM/TBST  Exposure Time: 37 seconds.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Immunoprecipitation - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Immunoprecipitation - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Histone H2A.X was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab229914 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab229914 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

    Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

    Lane 2: ab229914 IP in HeLa whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229914 in HeLa whole cell lysate

    Blocking and dilution buffer and concentration/ 5% NFDM/TBST.

    Exposure time/ 30 seconds.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Flow Cytometry - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Flow Cytometry - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/50 dilution(Red), compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human testis (PMID/ 24059746). The section was incubated with ab229914 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human breast carcinoma (PMID/ 27006338). The section was incubated with ab229914 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/100 dilution, followed by Ab229914 anti- Histone H2A.X ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Peptide Array - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Peptide Array - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    All batches of ab229914 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • ChIP - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    ChIP - Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

    Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.

    The ChIP was performed with 25 µg of chromatin, 5 µg of  ab229914 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20µl of Protein A/G sepharose beads.  The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

    Primers and probes are located in the first kb of the transcribed region.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab229914).

  • Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)
    Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade – BSA and Azide free (ab256544)

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