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 ab10799 (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.    

All lanes : Anti-Histone H3 antibody [mAbcam 10799] - ChIP Grade (ab10799) at 5 µg/ml

Lane 1 : Histone H1 Recombinant Protein
Lane 2 : Histone H2A Recombinant Protein
Lane 3 : Histone H2B Recombinant Protein
Lane 4 : Histone H3.1 Recombinant Protein
Lane 5 : Histone H4 Recombinant Protein

Lysates/proteins at 0.1 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa


Exposure time: 4 minutes

We recommend using 3% milk as the blocking agent in Western Blot.

Histone H3 [mAbcam 10799] - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Mouse monoclonal to and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab10799.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 15kDa; [mAbcam 10799] Histone H3 - ChIP Grade.

ab10799 staining human kidney sections by IHC-P using EXPOSE IHC detection kit (ab80436). Formalin fixed paraffin embedded tissue sections were pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab10799, 5µg/ml, for 1 hour at room temperature. DAB was used as the chromogen and the section was counterstained with haematoxylin and mounted with DPX.

All lanes : Anti-Histone H3 antibody [mAbcam 10799] - ChIP Grade (ab10799) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Lane 2 : Calf Thymus Histone preparation nuclear lysate at 0.5 µg
Lane 3 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat polyclonal to Mouse 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?


Exposure time: 90 seconds

IHC image of Histone H3 staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. 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 ab10799, 1µg/ml, for 8 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.