Immunocytochemistry - Histone H3 (tri methyl K4) antibody [mAbcam 1012] (ab1012)

SK-N-SK cells were fixed in 4% paraformaldehdye, permeabilized in 0.5% Triton X-100 and incubated with ab1012 (1/100). The antibody clearly stains the nucleus (red). The cells were counterstained with DAPI (blue). 100x magnification.

Darin McDonald, Cross Cancer Institute

ChIP - Histone H3 (tri methyl K4) antibody [mAbcam 1012] - ChIP Grade (ab1012)

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, 5 µg of  ab1012 (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.

Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)

ICC/IF image of ab1012 stained HeLa 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 (ab1012, 5µg/ml) overnight at +4ºC. 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) 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) HepG2, Hek293 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.

Western blot - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)

Lane 1 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012) at 1 µg/ml (Blocked in 5% BSA)
Lane 2 : Anti-Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012) at 1 µg/ml (Blocked in 5% MILK)

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate

Lysates/proteins at 0.5 µg per lane.

Secondary
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 : 10 minutes

Western blot - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)

All lanes : Anti-Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012) at 2 µg/ml

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

Lysates/proteins at 0.5 µg per lane.

Secondary
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

ELISA - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)

ELISA using ab1012 at varying antibody concentrations.

Curve_SPL4 indicates binding to the tri methyl K4 peptide ab1342. Curve 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.