Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab12209 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

Ab12209 staining Histone H3 (Tri Methyl K4) in Mouse hair follicle DSCs by ICC/IF (Immunocytochemistry/Immunofluorescence).  Cells were fixed with 3.7% paraformaldehyde, permeabilised with 0.2% Triton X-100 and blocked with 10% normal goat serum in PBS.  Samples were incubated with primary antibody at 1:200 dilution. An Alexa Fluor ^® 568 conjugated goat anti-mouse IgG was used as a secondary antibody.

All lanes : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 2 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (unmodified) peptide (ab7228) at 0.25 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.25 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.25 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.25 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.25 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.25 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.25 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.25 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.25 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.25 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 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?


Exposure time: 16 minutes

ICC/IF image of ab12209 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab12209, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

ELISA using ab12209 at varying antibody concentrations.

Curve_SPL4  indicates binding to the tri methyl K4 peptide ab1342. In addition, SPL3 indicates partial binding to the di methyl K4 peptide ab7768.  There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).

Binding to the following peptides was not seen:
SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.

Lane 1 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% BSA)
Lane 2 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 1 µg/ml (BLOCKED WITH 5% MILK)

All lanes : Calf Thymus Histone Preparation Nuclear Lysate

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 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?


Exposure time: 12 minutes

Overlay histogram showing HeLa cells stained with ab12209 (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 (ab12209, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.