Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab9050 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH (active) and MYO-D (inactive) promoters and over the γ-Actin gene (active).

Schematic diagram of the γ-Actin gene is shown on the top of the figure.

Black boxes represent exons and thin lines represent introns.

PCR products are depicted as bars under the gene.

ab9050 stained in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

Cells were fixed with 100% methanol for 5 minutes at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9050 at 0.1 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody (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.

All lanes : Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade (ab9050) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - unmodified K36 at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K36) peptide (ab1783) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K36) peptide (ab1784) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K36) peptide (ab1785) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K37) peptide (ab24417) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa

why is the actual band size different from the predicted?

ab9050 staining Histone H3 (tri-methyl K36) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab9050 at 0.1 µg/ml and ab7291 (anti beta-Tubulin) at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat anti-rabbit AlexaFluor^®488 secondary (ab150081) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor^®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

ab9050 staining Histone H3 (tri methyl K36) in Saos-2 (Human osteosarcoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton in PBS and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/1000) for 1 hour. An Alexa Fluor^®488-conjugated Goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

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