Synthetic peptide within Histone H4 aa 1-100 (symmetric di methyl R3) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
(Peptide available as
Our Abpromise guarantee covers the use of ab5823 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|PepArr||Use a concentration of 0.2 - 0.02 µg/ml.|
|ChIP||Use 2-4 µg for 25 µg of chromatin.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 14 kDa (predicted molecular weight: 11.5 kDa).Can be blocked with Human Histone H4 (symmetric di methyl R3) peptide (ab14791).|
This blot was produced using a 4-12% 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 2% Bovine Serum Albumin before being incubated with ab5823 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
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 ab5823 (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.
IHC image of ab5823 staining Histone H4 (symmetric di methyl R3) 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 ab5823, 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.
All batches of ab5823 are tested in Peptide Array against peptides to different Histone H4 and H2A 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 H4 - symmetric di methyl R3 peptide (ab14791), indicating that this antibody specifically recognises the Histone H4 - symmetric di methyl R3 modification.
ab14791 - Histone H4 - symmetric di methyl R3
ab17416 - Histone H4 asymmetric di methyl R3
ab15821 - Histone H4 - unmodified
ab22399 - Histone H2A symmetric di methyl R3
ab22398 - Histone H2A asymmetric di methyl R3
ab13186 - Histone H2A - unmodified