Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: HDAC2 knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab16032 observed at 60 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab16032 detected the expected band for HDAC2 in wild-type HAP1 cells and the band was not seen in HDAC2 knockout HAP1 cells. Additional cross-reactive bands were detected. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  Ab16032 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

ab16032 staining HDAC2 in wild-type HAP1 cells (top panel) and HDAC2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16032 at 0.5μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ICC/IF image of ab16032 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 ab16032 at 5µg/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.

All lanes : Anti-HDAC2 antibody (ab16032) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 55.3 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?
Additional bands at: 50 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

HDAC2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5ug of Rabbit polyclonal to HDAC2 and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extractdiluted in RIPA buffer was added to each sample and incubated for 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 ab16032. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 60ka: HDAC2.