Anti-Histone H3 (symmetric di methyl R8) antibody (ab130740) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.25 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes


This blot was produced using a 10% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab130740 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

IHC image of Histone H3 (symmetric di methyl R8) staining in Human Skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab130740, 1µg/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.

All batches of ab130740 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - symmetric di methyl R8 peptide (ab154299), indicating that this antibody specifically recognises the Histone H3 - symmetric di methyl R8 modification.

1. ab154299 - Histone H3 - symmetric di methyl R8
2. ab154295 - Histone H3 - asymmetric di methyl R8
3. ab154298 - Histone H3 - mono methyl R8
4. ab7228 - Histone H3 - unmodified
5. ab154465 - Histone H3 - symmetric di methyl R2
6. ab154466 - Histone H3 - asymmetric di methyl R2
7. ab154425 - Histone H3 - symmetric di methyl R17
8. ab16935 - Histone H3 - asymmetric di methyl R17
9. ab154427 - Histone H3 - symmetric di methyl R26
10. ab2854 - Histone H3 - asymmetric di methyl R26

IHC image of ab130740 staining Histone H3 (symmetric di methyl R8) in human kidney 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 ab130740, 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.

Histone H3 was immunoprecipitated using 0.5mg Calf thymus histone whole cell extract, 5µg of Rabbit polyclonal to Histone H3 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, Calf thymus histone 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 ab130740.

Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.

Band: 15kDa; Histone H3