Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

• ICC/IF

Ap

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

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.

• ICC/IF

AbReview22136****

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

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.

This image is courtesy of an anonymous Abreview

• ChIP

Unknown

ChIP - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

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.

• ICC/IF

AbReview34550****

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

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.

This image is courtesy of an Abreview submitted by Kirk Mcmanus.

• WB

AbReview16648****

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

All lanes:

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1/1000 dilution

Lane 1:

Nuclear lysates prepared from Hela cell: SiRNA untreated at 10 µg

Lane 2:

Nuclear lysates prepared from Hela cells treated with G9a SiRNA at 10 µg

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

false

Exposure time: 10min

This image is courtesy of an anonymous abreview.

• WB

AbReview27649****

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

All lanes:

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1/300 dilution

All lanes:

HUH-7 cells at 15 µg

Secondary

All lanes:

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

Predicted band size: 15 kDa

false

This image is courtesy of an anonymous Abreview

• WB

AbReview39017****

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

All lanes:

Diluent: TBS + 0.1% Tween 20 + 5% BSA. Incubated for 12 hours at 4°C

All lanes:

HeLa whole cell lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 15 kDa

false

Exposure time: 1min

This image is courtesy of an anonymous Abreview

• WB

Project

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (AB8896)

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.

All lanes:

Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab8896) at 1 µg/mL

Lane 1:

Calf Thymus Histone Preparation at 0.5 µg

Lane 2:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (mono methyl K9) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-mono-methyl-k9-peptide-ab1771'>ab1771</a>)

Lane 3:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (di methyl K9) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-di-methyl-k9-peptide-ab1772'>ab1772</a>)

Lane 4:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (tri methyl K9) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-tri-methyl-k9-peptide-ab1773'>ab1773</a>)

Lane 5:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (mono methyl K27) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-mono-methyl-k27-peptide-ab1780'>ab1780</a>)

Lane 6:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (di methyl K27) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-di-methyl-k27-peptide-ab1781'>ab1781</a>)

Lane 7:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (tri methyl K27) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-tri-methyl-k27-peptide-ab1782'>ab1782</a>)

Lane 8:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (mono methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-mono-methyl-k4-peptide-ab1340'>ab1340</a>)

Lane 9:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (unmodified) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-unmodified-peptide-ab7228'>ab7228</a>)

Lane 10:

Calf Thymus Histone Preparation at 0.5 µg with Human Histone H3 (unmodified ) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-unmodified-peptide-ab2623'>ab2623</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

false