Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab9045 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

Anti-mono methyl lysine 9 of histone H3 (green) has a distribution often associated with euchromatic probes (small foci).  Most of these foci localize to regions that contain obvious enrichments of DNA with DAPI staining (red).  The perinucleolar chromatin is typically a site enriched in monomethylated lysine 9. 

Top left: Mono-methyl Lys 9 (ab9045); Bottom left: DAPI; Top right: Merge of ab9045 (green) and DAPI (red).

All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

Lane 2 : Human Histone H3 (unmodified) peptide (ab7228)
Lane 3 : Human Histone H3 (mono methyl K27) peptide (ab1780)
Lane 4 : Human Histone H3 (di methyl K27) peptide (ab1781)
Lane 5 : Human Histone H3 (tri methyl K27) peptide (ab1782)
Lane 6 : Human Histone H3 (mono methyl K4) peptide (ab1340)
Lane 7 : Human Histone H3 (mono methyl K9) peptide (ab1771)

Predicted band size: 17 kDa

Rabbit polyclonal to Histone H3 K9 Methyl K9 (1/1000)

Peptides at 1 ug/ml

1XTBS, 5%BSA, 0.5% Tween

This antibody shows significantly greater reactivity with mono methyl K9. This can be seen in lane 7, as the addition of ab1771 (mono methyl K9) completely blocks the activity of ab9045. Weaker cross-reactivity is seen against mono methyl K27. This is shown in lane 3, as the addition of ab1780 only partially blocks the activity of ab9045.

All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (unmodified) peptide (ab7228) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 17 kDa

ab9045 staining Histone H3 (mono methyl K9) in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Triton for 1h. The cells were then incubated with the antibody ab9045 at 0.1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse 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.

Histone H3 (mono methyl K9) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K9) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9045.
Secondary: Anti-rabbit IgG VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution.
Band: 17kDa: Histone H3 (mono methyl K9).