ab24684 staining Histone H3 (di methyl K27) in HeLa cells. The cells were fixed with 100% Methanol (5min), permeabilized with 0.1%PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab24684 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

 

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of Histone H3 (di methyl K27) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab24684, 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.

All lanes : Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684) at 1 µg/ml

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Human Histone H3 peptide (ab17163) at 0.5 µg/ml
Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 4 : Calf thymus histone lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
Lane 6 : Calf thymus histone lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 7 : Calf thymus histone lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 8 : Calf thymus histone lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
Lane 9 : Calf thymus histone lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
Lane 10 : Calf thymus histone lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
Lane 11 : Calf thymus histone lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

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

Predicted band size: 15.2 kDa
Observed band size: 17 kDa

why is the actual band size different from the predicted?


Exposure time: 5 seconds

The peptide competition assay shows that this antibody is very specific for Histone H3 di-methylated at K27. It does not recognise Histone H3 mono, di, or tri methylated at K4, Histone H3 mono, di, or tri methylated at K9, or Histone H3 mono, or tri methylated at K27.

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab24684 (blue), and 20µl 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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

Immunocytochemistry/ Immunofluorescence analysis of IMR32, Human Neuroblatoma cells labeling Histone H3 (di methyl K27) with ab24684 at 1/100 dilution. The cells were fixed with paraformaldehyde and permeabilized with 0.3% triton X 100. The cells were blocked with 1% BSA for 1 hour at 25°C, followed by incubation with primary antibody in 1% BSA containing TBST for 16 hours at 4°C. A Goat Anti Rabbit-IgG Cy3^® was used as the secondary antibody at 1/1000 dilution.

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