All lanes : Anti-Histone H3 (phospho T3) antibody [EP1702Y] (ab78351) at 1/500000 dilution

Lane 1 : HeLa cell lysates
Lane 2 : HeLa cell lysates treated with FBS + Calyculin A.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 16 kDa
Observed band size: 14 kDa why is the actual band size different from the predicted?

ab78351 at 1/100 dilution staining Histone H3 in human breast carcinoma tissue, using a HRP/AP polymerized secondary antibody.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunocytochemistry analysis of HeLa (human cervix adenocarcinoma epithelial cell) labeling Histone H3 with purified ab78351 at 1/250 dilution. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.18 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

Overlay histogram showing HeLa cells stained with ab78351 (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 (ab78351, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.