IHC image of Histone H3 (phospho T11) staining in human normal testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. 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 ab5168, 1µg/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.

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.

Anti-Histone H3 (phospho T11) antibody (ab5168) at 1/500 dilution + Calf thymus histone lysate

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/2000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Rabbit polyclonal to Histone H3 (phospho T11) - ab5168 at 1/500 dilution.

Lane 1 contains 1ug calf thymus histone prep.

Secondary ab: Goat anti-rabbit IgG HRP conjugate ab6721 (1/2000)

 

SKN-SH cells were fixed in 4% paraformaldehyde for 10 mins, permeabilized in PBS-0.5% Triton X-100 for 5 mins and incubated for 30 minutes with ab5168 (1/100). The slides were rinsed once in PBS-Triton (0.1%), twice in PBS then incubated with the secondary antibody for 30 mins. The DNA is stained with DAPI (blue). Clear nuclear staining with ab5168 can be seen (green). 100x magnification.

All lanes : Anti-Histone H3 (phospho T11) antibody (ab5168) at 1 µg/ml

Lane 1 : Colcemid treated HeLa Histone prep at 5 µg
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg
Lane 3 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (phospho T11) peptide (ab24444) at 1 µg/ml
Lane 4 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (phospho T11) peptide (ab24444) at 1 µg/ml
Lane 5 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml
Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml
Lane 7 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 8 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml

Secondary
All lanes : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

This antibody is blocked by the Histone H3 phospho T11 peptied but not by the respective unmodified peptide or the Histone H3 phospho S10 peptide. This confirms that the antibody is specific for phospho T11 of Histone H3.

ICC/IF image of ab5168 stained Hek293 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5168, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa and HepG2 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.