All lanes : Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution

All lanes : Human brain tissue

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP secondary antibody (HRP) at 1/1000 dilution
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 15 kDa
Observed band size: 16,25 kDa why is the actual band size different from the predicted?

This blot was developed using a higher sensitivity ECL substrate.

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.

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.

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.

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.

*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol.

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.

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.

 

Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution + HEK-293 (human embryonic kidney epithelial cell), histone extract at 20 µg

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

Predicted band size: 15 kDa
Observed band size: 16, 25 kDa why is the actual band size different from the predicted?

A 25-kDa band, likely to be ubiquitinated H2A.X, is observed. The molecular weights are consistent with what have been described in the literature (PMID: 24603765)

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

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)

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Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.