Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with 0.75% formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab177840 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the first kb of the transcribed region.

All lanes : Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution

Lanes 1 & 6 : Histone H1 Recombinant Protein
Lanes 2 & 7 : Histone H2A Recombinant Protein
Lanes 3 & 8 : Histone H2B Recombinant Protein
Lanes 4 & 9 : Histone H3.1 Recombinant Protein
Lanes 5 & 10 : Histone H4 Recombinant Protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 11 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

Lanes 1-5: 1% BSA blocking buffer

Lanes 6-10: 3% Milk blocking buffer

 

We recommend using 3% milk as the blocking agent for Western blot.

Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution + Drosophila whole lysates at 10 µg

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 11 kDa
Observed band size: 11 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/5000 dilution

Lane 1 : HeLa whole cell lysates
Lane 2 : NIH/3T3 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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 11 kDa
Observed band size: 11 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

ab177840 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Histone H4 with ab177840 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab177840 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin. 

Negative control: PBS instead of primary antibody; secondary antibody is prediluted 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 pancreas tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of mouse pancreas tissue is observed. Counter stained with Hematoxylin.

Negative control: PBS instead of primary antibody; secondary antibody is prediluted 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 cerebral cortex tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on neuron cells of cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Negative control: PBS instead of primary antibody; secondary antibody is prediluted 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.