Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab108245 (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.
All batches of ab108245 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - tri methyl K27 peptide (ab1782), indicating that this antibody specifically recognises the Histone H3 - tri methyl K27 modification.ab7228 - Histone H3 - unmodifiedab1340 - Histone H3 - mono methyl K4ab7768 - Histone H3 - di methyl K4ab1342 - Histone H3 - tri methyl K4ab1771 - Histone H3 - mono methyl K9ab1772 - Histone H3 - di methyl K9ab1773 - Histone H3 - tri methyl K9ab1780 - Histone H3 - mono methyl K27ab1781 - Histone H3 - di methyl K27ab1782 - Histone H3 - tri methyl K27
All lanes : Anti-Histone H3 (tri methyl K27) antibody - ChIP Grade (ab108245) at 1 µg/mlLane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Immunising peptide at 1 µg/mlLane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Non modified peptide at 1 µg/mlLane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 1 µg/mlLane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/mlLane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 1 µg/mlLane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 1 µg/mlLane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Hum
ICC/IF image of ab108245 stained HeLa cells. The cells were 100% Methanol fixed (5 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 (ab108245, 0.2µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 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.