All lanes : Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab7766) at 1 µg/ml

Lane 1 : HeLa nuclear lysate (triton enriched)
Lane 2 : NIH 3T3 nuclear lysate (triton enriched)
Lane 3 : PC12 nuclear lysate (triton enriched

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Blocking buffer: 2% BSA

Gel type: MES

Exposure Time: 1 minute

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 ab7766 (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 ab7766 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 - di methyl K4 peptide (ab7768), indicating that this antibody specifically recognises the Histone H3 - di methyl K4 modification.

1. ab1340 - Histone H3 - mono methyl K4
2. ab1342 - Histone H3 - tri methyl K4
3. ab1771 - Histone H3 - mono methyl K9
4. ab1772 - Histone H3 - di methyl K9
5. ab1773 - Histone H3 - tri methyl K9
6. ab1780 - Histone H3 - mono methyl K27
7. ab1781 - Histone H3 - di methyl K27
8. ab1782 - Histone H3 - tri methyl K27
9. ab7228 - Histone H3 - unmodified
10. ab7768 - Histone H3 - di methyl K4

ab7766 stained in HeLa cells. Cells were fixed with 100% methanol (5 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 ab7766 at 0.2 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Dimethylated lysine 4 (green) is found in several hundred small nuclear foci that do not colocalize with condensed regions of chromatin (DAPI stained, red).  The perinuclear and perinucleolar heterochromatin do not stain with this antibody.

Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab7766) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Additional bands at: 14 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

ab7766 at 1/200 staining human U2OS (osteosarcoma) cells by ICC/IF. The cells were paraformaldehyde fixed and then stained with the antibody for 1 hour. A Cy2 ® conjugated donkey anti-rabbit antibody was used as the secondary (green). The image shows uniformal staining of the whole nucleus, with several specles found. The insert shows H3-di methyl K4 of tw2 cells only. DAPI nuclear staining is shown in blue.

See Abreview

MCF7 cells were incubated at 37&degC for 24h with vehicle control (0 &microM) and different concentrations of tranylcypromine hydrochloride (ab120606). Increased expression of Histone 3 K4 di-methyl (ab7766) in MCF7 cells correlates with an increase in tranylcypromine hydrochloride concentration, as described in literature.

Nuclear extracts were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10&microg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab7766 at 1 &microg /ml and ab1791 at 1 &microg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody (ab97051) at 1/10000 dilution and visualised using ECL development solution.