ab9052 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9052 at 0.5µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

Histone H4 (di methyl K20) was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to Histone H4 (di methyl K20) 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9052.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 14kDa: Histone H4 (di methyl K20).

All lanes : Anti-Histone H4 (di methyl K20) antibody (ab9052) at 1/2000 dilution

Lane 1 : yH4 17-24 peptide
Lane 2 : Unmodified yH4 17-24 peptide
Lane 3 : Mono methyl K20 yH4 17-24 peptide
Lane 4 : Di methyl K20 yH4 17-24 peptide
Lane 5 : Tri methyl K20 yH4 17-24 peptide

Blocking peptides at 1 µg/ml per lane.

Performed under reducing conditions.

All lanes : Upper blot - Histone H4 antibody (gift of A. Verreault)

Lower blot - Rabbit polyclonal to Histone H4 di methyl K20 (ab9052) at 1/2000

Lane 1 : S.cerevisiae extract
Lane 2 : S.pombe extract
Lane 3 : S.pombe rH4
Lane 4 : Drosophila rH4
Lane 5 : Histones (Roche)

Di methylation at K20 is seen in S. pombe and in calf thymus (Roche). S. cerevisiae lacks K20 (di) methylation.

IHC image of ab9052 staining in human pancreas adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab9052, 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.

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.