Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)

ICC/IF image of ab9052 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9052 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM 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).

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