Immunoprecipitation dilution was 1/60.

Western blot was performed on the immunoprecipitate using ab32388 at 1/500 dilution (2.444 μg/ml). VeriBlot for IP secondary antibody (HRP) (ab131366) at 1:1000 dilution.

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

All lanes :

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5µM Nocodazole for 24h whole cell lysate at 10 µg
Lane 2 : ab32388 IP in Nocodazole treated HeLa whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab32388 in nocodazole treated HeLa whole cell lysate

Observed band size: 17 kDa why is the actual band size different from the predicted?

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 ab32388 (red), and 20 µl of Protein A/G sepharose beads. Rabbit normal IgG was added to the beads control (gray). 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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, LP starved and non-starved, labeling anti-Histone H3 (phospho S28) with Ab32388 at 1/100 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor®488 (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on M phase of HeLa cells, then the signal decreased after LP treatment.

For the pan antibody, there was no great difference after LP treatment. The data showed mostly nuclear staining

All lanes : Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab32388) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Whole cell lysate from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 100ng/ml nocodazole for 18 hours
Lane 3 : Whole cell lysate from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 100ng/ml nocodazole for 18 hours. Membrane incubated with phosphatase

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 17 kDa
Observed band size: 17 kDa


Exposure time: 15 seconds

Blocking/dilution buffer: 2% BSA/TBST

Dot blot performed using ab32388 at a dilution of 1/100. Lane 1 - Unmodified H3 peptide. Lane 2 - H3S28ph peptide. Lane 3 - H3.3S28ph peptide. Lane 4 - H3.3S31ph peptide. A HRP conjugated goat anti-rabbit (H+L) was used as the secondary antibody at a dilution of 1/2500. The exposure time was 3 minutes and the dilution and blocking buffer used were 5% NFDM/TBST.

All lanes : Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab32388) at 1/2000 dilution

Lane 1 : NIH 3T3 cell lysate -untreated
Lane 2 : NIH 3T3 cell lysate -treated with FBS + CalA.

Predicted band size: 17 kDa
Observed band size: 17 kDa

IHC image of ab32388 staining Histone H3 in Human normal colon formalin fixed paraffin embedded tissue* sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32388, 0.1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Overlay histogram showing HeLa cells stained with ab32388 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32388, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Overlay histogram showing HeLa cells stained with ab32388 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 90% methanol. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32388, 1:1200 dilution, 1.02 μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1:2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.