All lanes : Anti-HDAC1 antibody (ab19845) at 1 µg/mlLane 1 : HeLa whole cell lysateLane 2 : HeLa whole cell lysate with Human HDAC1 peptide (ab20434) at 1 µg/mlLysates/proteins at 20 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab7090) at 1/5000 dilution

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-iTTM 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.

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 BondTM 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 70oC; 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.