Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab177863(red), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

All lanes : Anti-Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) antibody [EPR17589] - ChIP Grade (ab177863) at 1/4000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysates
Lane 2 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysates
Lane 4 : Untreated NIH/3T3 (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 14,15 kDa
Observed band size: 14,15 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cell line labeling Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) with ab177863 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cell line. Acetylation level increased after treatment with Trichostatin A (500 ng/ml) for 4 hours. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab177863 at 1/400 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution.

ab177863 was 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.

All lanes : Anti-Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) antibody [EPR17589] - ChIP Grade (ab177863) at 1/4000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line, treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysate.
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line, treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysate. with H2AK5ac
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line, treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysate. with H2A.XK5ac
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line, treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysate. with Unmodified H2A.X
Lane 5 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line, treated with 500 ng/ml Trichostatin A for 4 hours. Whole cell lysate. with Unmodified H2A

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 14,15 kDa
Observed band size: 14,15 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) with ab177863 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on human colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) with ab177863 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H2A.X (acetyl K5) + Histone H2A (acetyl K5) with ab177863 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on rat colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.