Confocal microscopy of sagittal sections through the rat pineal gland (PG) immunolabeled for Pax6 (green) and the mitotic M-phase marker phospho-histone H3 (PH3, ab14955, red). All PH3+ mitotic cells expressed Pax6; Pax6 levels, however, varied among dividing pinealocyte precursor cells. The number of mitotic precursor cells increased progressively during embryogenesis, and dropped quickly after birth. White arrowheads: Pax6^high mitoses. White arrows: Pax6^low mitoses.

E, embryonic day. PC, posterior commissure. P, postnatal day. SCO, subcommissural organ. 3v, third ventricle.

All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] (ab14955) at 1 µg/ml

Lane 1 : Control HeLa Histone Prep 0.5ug
Lane 2 : Colcemid treated HeLa Histone prep 0.5ug
Lane 3 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (tri methyl K9, phospho S10) peptide (ab15644) at 1 µg/ml
Lane 4 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (tri methyl K9, phospho S10) peptide (ab15644) at 1 µg/ml
Lane 5 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (unmodified) peptide (ab7228) at 1 µg/ml
Lane 6 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (unmodified) peptide (ab7228) at 1 µg/ml
Lane 7 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 8 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 9 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/ml
Lane 10 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/ml

Secondary
All lanes : Rabbit polyclonal to Mouse IgG H&L (HRP) at 1/5000 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?

IHC image of ab14955 staining Histone H3 (phospho S10) in human kidney 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 (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab14955, 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 hematoxylin 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.

By ELISA, ab14955 detects:
the singly modified phospho S10 peptide and the dual modified phospho S10 and tri methyl K9 peptide (the orange and grey lines respectively),
less strongly detects the phospho S28 peptide (purple line),
does not detect the equivalent non-modified Histone H3 peptide for S10 or the singly modified tri methyl K9 peptide (the 2 lines at the bottom of the figure).

The antibody recognises phospho S28 by ELISA (although a phospho S28 peptide does not block the antibody in Western blotting).

All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] (ab14955) at 1/2000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : HeLa cell lysate treated with calyculin A
Lane 3 : HeLa cell lysate treated with calyculin A and alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/20000 dilution

Predicted band size: 15 kDa


Exposure time: 1 second

All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] (ab14955) at 0.5 µg/ml

Lane 1 : Control HeLa Histone prep
Lane 2 : Colecemid treated HeLa Histone prep
Lane 3 : Control HeLa Histone prep with Human Histone H3 (unmodified) peptide (ab7228) at 1 µg/ml
Lane 4 : Colecemid treated HeLa Histone prep with Human Histone H3 (unmodified) peptide (ab7228) at 1 µg/ml
Lane 5 : Control HeLa Histone prep with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/ml
Lane 6 : Colecemid treated HeLa Histone prep with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/ml
Lane 7 : Control HeLa Histone prep with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 8 : Colecemid treated HeLa Histone prep with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml

Lysates/proteins at 5 µg per lane.

Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

Predicted band size: 15 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] (ab14955) at 1/1000 dilution

Lane 1 : Wild type 0-4 hour old fruit fly embryo lysate
Lane 2 : 0-4 hour old fruit fly embryo lysate expressing wee RNAi

Secondary
All lanes : Anti-mouse IgG, peroxidase-linked at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa


Exposure time: 5 seconds

Blocking: 10% BSA

wee shRNA embryos (lane 2) should display elevated phospho H3Ser10 levels relative to wild type

See Abreview