Western blot - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
ab8895 is specific for mono-methylated Lysine 4 of histone H3 and does not recognize di- or tri-methyl Lysine 4 nor methylation at Lysine 9. This is shown in lane 2 where the activity of the antibody is specifically blocked by the addition of the immunizing peptide (ab1340).
All lanes:
Western blot - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895) at 1/500 dilution
All lanes:
Calf thymus histone lysate
Secondary
All lanes:
Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 18 kDa
Exposure time: 2min
Western blot - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
Blocking buffer: 2% BSA
Gel type: MES
All lanes:
Western blot - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895) at 1 µg/mL
Lane 1:
Calf Thymus Histone at 0.5 µg
Lane 2:
HeLa Nuclear – Triton Prep at 10 µg
Lane 3:
NIH3T3 Nuclear – Triton Prep at 10 µg
Lane 4:
PC12 Nuclear at 10 µg
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
Exposure time: 30s
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
ab8895 staining Histone H3 (mono methyl K4) in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 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 ab8895 at 1µg/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 100% methanol (5 min).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
IHC image of ab8895 staining Histone H3 (mono methyl K4) in 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
- for 20 mins. The section was then incubated with ab8895, 0.5μg/ml, 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.
ChIP - Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)
Chromatin was prepared from U-2 OS 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 ab8895 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH and ALDOA (active) and MYO-D (inactive) promoters and over the y-Actin gene (active). Schematic diagram of the y-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.
