Synthetic peptide, corresponding to amino acids 473-488 of Human HDAC2
This monoclonal antibody to HDAC2 has been knockout validated in Western blot. The expected band for HDAC2 was observed in wild type cells and the band was not seen in HDAC2 knockout cells.
Alternative versions available:
Our Abpromise guarantee covers the use of ab51832 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|IHC-P||Use at an assay dependent concentration.|
|WB||1/1000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa).|
|IP||Use at an assay dependent concentration.
Use 10ug for 10-50ug cell lysate.
|ChIP||Use at an assay dependent concentration.
Use 10ug for 25ug of DNA
|ICC/IF||1/25 - 1/100.|
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: HDAC2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: A431 whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab51832 observed at 60 kDa. Red - loading control, ab129002, observed at 130 kDa.
ab51832 detected the expected band for HDAC2 in wild-type HAP1 cells and the band was not seen in HDAC2 knockout HAP1 cells. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab51832 and ab129002 (Rabbit anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
ab51832 stained MCF7 cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51832 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour 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.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 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 ab51832 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
Chromatin was prepared from the cell lysate of rat liver, cultured hepatoma cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The primary antibody was diluted 1/100 and incubated with the sample for 1.5 hours at 25°C. Anti RNA polymerase II was used on the positive control, whilst normal mouse IgG was used in the negative control, to compare difference of HDAC2 binding on silencer DNA and promoter DNA of a gene. Data is normalized to 10% Input. The immunoprecipitated DNA was quantified by real time PCR.