ab78517 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab78517 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

All lanes : Anti-Histone H4 (di methyl K20, tri methyl K20) antibody [6F8-D9] (ab78517) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H4 peptide (ab14963) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H4 (mono methyl K20) peptide (ab17043) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H4 peptide (17-24) - di methyl K20 at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H4 (tri methyl K20) peptide (ab17567) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 11 kDa
Observed band size: 13 kDa

why is the actual band size different from the predicted?


Exposure time: 90 seconds


ab78517 is specific for Histone H4 (tri-methyl K20). This is illustrated in lane 8 where the activity of ab78517 is specifically blocked by the addition of the immunizing peptide (ab17567).

ICC/IF image of ab78517 stained mouse oocyte. Oocytes were fixed in formaldehyde and then incubated in 3% serum with 0.25% Triton for 30 minutes at 37°C. Cells were incubated with the antibody (ab78517) at 1/200 for 2 hours at 37°C. The secondary antibody used was a polyclonal Goat-anti-mouse conjugated to Alexa Fluor® 488 (1/250).

See Abreview

IHC image of Histone H4 (tri methyl K20) staining in normal human skin 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 ab78517, 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.

Overlay histogram showing HeLa cells stained with ab78517 (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. The cells were then incubated with the antibody (ab78517, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse 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.