Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: HDAC1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human breast carcinoma lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab19845 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab19845 was shown to recognize HDAC1 when HDAC1 knockout samples were used, along with additional cross-reactive bands. Wild-type and HDAC1 knockout samples were subjected to SDS-PAGE. ab19845 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

ab19845 staining HDAC1 in wild-type HAP1 cells (top panel) and HDAC1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 ab19845 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).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: HDAC1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human breast carcinoma lysate (20 µg) or K562 lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab19845 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab19845 and a competitor's top cited rabbit polyclonal antibody.

ICC/IF image of ab19845 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab19845, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Panel A shows localisation of ab19845 to the nuclei, Panel B has the Alexa Fluor® 488 channel removed for comparison.

The image shows staining of human tonsil tissue using ab19845. Staining was nuclear and was equally successful using Tris EDTA pH9 or Citrate pH6 for antigen retrieval. Staining was prevalent in almost all cellular compartments of the tonsil.

All lanes : Anti-HDAC1 antibody (ab19845) at 1 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : HeLa whole cell lysate with Human HDAC1 peptide (ab20434) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab7090) at 1/5000 dilution

Predicted band size: 55 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

ab19845 at a 1/3000 dilution staining asynchronous HeLa cells by ICC/IF. The cells were paraformaldehyde fixed and immunofluorescently labelled with ab19845 for 30 minutes at room temperature. Bound antibody was detected using a Cy3 conjugated goat anti-rabbit antibody. Nuclei were visuallised using DAPI staining. The antibody was found to be highly enriched in the nucleus.

This image is courtesy of an Abreview submitted by Kirk McManus.

See Abreview

ICC/IF image of ab19845 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19845, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 cells at 1µg/ml, and in 100% methanol fixed (5 min) MCF7 and HepG2 cells at 1µg/ml

IHC image of HDAC1 staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab19845, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX

HDAC1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to HDAC1 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 ab19845.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 60ka: HDAC1.