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

• ChIP

Unknown

ChIP - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (AB8898)

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 ab8898 (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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (AB8898)

ICC/IF image of ab8898 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 (ab8898, 0.1μg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (AB8898)

IHC image of ab8898 staining Histone H3 (tri methyl K9) in normal human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8898, 1/400 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (AB8898)

ab8898 staining Histone H3 (tri methyl K9) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab8898 at 0.5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (AB8898)

Lane 8 shows that Rabbit polyclonal to Histone H3 (tri methyl K9) is blocked by the addition of the immunizing peptide (ab1773). Cross-reactivity with Histone H3 peptide - tri methyl K27 (ab1782) is also shown in Lane 11.

All lanes:

Western blot - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1 µg/mL

Lane 1:

Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Lane 2:

Calf Thymus Histone Preparation Nuclear Lysate 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 3:

Calf Thymus Histone Preparation Nuclear Lysate 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 4:

Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H3 (di methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-di-methyl-k4-peptide-ab7768'>ab7768</a>)

Lane 5:

Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H3 (tri methyl K4) peptide (<a href='/en-us/products/proteins-peptides/human-histone-h3-tri-methyl-k4-peptide-ab1342'>ab1342</a>)

Lane 6:

Calf Thymus Histone Preparation Nuclear Lysate 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 7:

Calf Thymus Histone Preparation Nuclear Lysate 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 8:

Calf Thymus Histone Preparation Nuclear Lysate 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 9:

Calf Thymus Histone Preparation Nuclear Lysate 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 10:

Calf Thymus Histone Preparation Nuclear Lysate 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 11:

Calf Thymus Histone Preparation Nuclear Lysate 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>)

Secondary

All lanes:

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

Predicted band size: 15 kDa

Observed band size: 17 kDa

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