Anti-HDAC3 antibody - ChIP Grade (ab7030)

  ChIP - HDAC3 antibody - ChIP Grade (ab7030)

Sonicated Chromatin prepared from untreated (UI) or 17beta-estradiol (E2) treated MCF7 cells was subjected to the ChIP procedure with ab7030 to HDAC3 and the immunoprecipitated chromatin was analysed in the proximal region of the estrogen-responsive pS2 promoter (as shown above) and quantified by real-time PCR (values are nomalized over inputs). The primers are designed to follow the nucleosome E (including the Estrogen Responsive Element ERE). 2 µl of ab7030 and 2x106 cells were used in each ChIP experiment.

Sylvain Daujat

  Immunofluorescence - HDAC3 antibody - ChIP Grade (ab7030)

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 1/5000 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 ab7030, blue by DAPI. A small amount of cytoplasmic staining was seen with ab7030.

Michael Mancini, Baylor College of Medicine

  Western blot - HDAC3 antibody - ChIP Grade (ab7030)

All lanes : Anti-HDAC3 antibody - ChIP Grade (ab7030) at 1/10000 dilution

Lane 1 : Whole cell lysate from human HEK293 cell line
Lane 2 : Whole cell lysate from human HEK293 cell line treated with HDAC3 gene silencing shRNA

Lysates/proteins at 20 µg per lane.

Secondary
HRP-conjugated goat anti-rabbit Ig at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 49 kDa


Exposure time : 10 seconds

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