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  ab8896 (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.

Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1/300 dilution (Incubated for 10 minutes at 20°C. Diluent buffer used was 0.5% milk in TBS, 0.1% Tween.) + HUH-7 cells at 15 µg

Secondary
Sheep anti-rabbit IgG (HRP) at 1/20000 dilution

Predicted band size: 15 kDa

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All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation
Lane 2 : Calf Thymus Histone Preparation with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation with Human Histone H3 (unmodified) peptide (ab7228) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation with Human Histone H3 (unmodified ) peptide (ab2623) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 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?

ab8896 specifically recognises mono-methyl K9 (lane 1) in calf thymus lysate at 17kDa. ab8896 is successfully blocked using the immunizing peptide (lane 2 ab1771), but not the di-methyl K9 (lane 3 ab1772), the tri-methyl K9 (lane 4 ab1773), the mono-methyl K27 (lane 5 ab1780), the di-methyl K27 (lane 6 ab1781), the tri-methyl K27 (lane 7 ab1782), the mono-methyl K4 (lane 8 ab1340) nor the corresponding unmodified peptides (lane 9 ab7228, lane 10 ab2623). This implies that ab8896 is specific to mono-methyl K9.

Ab8896 staining Histone H3 (mono methyl K9) in HeLa (Cervical cancer cell line) cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton/TBS and blocked with 2% serum for 30 minutes at 25°C. Samples were incubated with primary antibody at 1/100 dilution for 1 hour at 25°C. An Alexa Fluor^® 488-conjugated Goat anti-rabbit was used as a secondary antibody at a 1/1000 dilution.

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Diluent: TBS + 0.1% Tween 20 + 5% BSA. Incubated for 12 hours at 4°C + HeLa whole cell lysates at 20 µg

Secondary
Anti-rabbit IgG, HRP-linked Antibody at 1/2000 dilution

Predicted band size: 15 kDa


Exposure time: 1 minute

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Ab8896 staining Histone H3 (mono methyl K9) in HeLa cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.05% Triton X100 in PBS. Samples were incubated with primary antibody at 1/200 dilution in PBS for 1 hour at 22°C. An Alexa Fluor^® 488-conjugated Goat anti-rabbit was used as the secondary antibody at a 1/200 dilution.

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ICC/IF image of ab8896 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 ab8896 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was a goat anti-rabbit Alexa Fluor® 488 (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1/1000 dilution

Lane 1 : Nuclear lysates prepared from Hela cell: SiRNA untreated
Lane 2 : Nuclear lysates prepared from Hela cells treated with G9a SiRNA

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Alkaline Phosphatase conjugated rabbit polyclonal rabbit IgG (Fc) at 1/5000 dilution

Predicted band size: 15 kDa
Observed band size: 16 kDa why is the actual band size different from the predicted?


Exposure time: 10 minutes