Immunofluorescence - HDAC2 antibody - ChIP Grade (ab7029)

HeLa cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/600 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab7029, blue staining by DAPI.

Michael Mancini, Baylor College of Medicine

ChIP - HDAC2 antibody - ChIP Grade (ab7029)

Chromatin was prepared from mouse skin epidermis whole tissue lysate.  Cells were fixed with 1% fromaldehyde for 10 minutes.  Wild type animal samples were incubated with rabbit IgG or ab7029 (diluted 1/800) for 12 hours at 4°C and the immunoprecipitated DNA was amplified using primers flanking the proximal promoter region and quantified by semiquantitative PCR.  Rabbit IgG did not produce significant amplification.

This image is courtesy of an anonymous Abreview

Immunoprecipitation - Anti-HDAC2 antibody - ChIP Grade (ab7029)

HDAC2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to HDAC2 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 ab7029.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: Band: 60ka: HDAC2; 55kDa; Heavy Chain.