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Synthetic peptide conjugated to KLH derived from within residues 450 to the C-terminus of Human HDAC1.
(Peptide available as ab20434.)
Our Abpromise guarantee covers the use of ab19845 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-FrFl||Use at an assay dependent concentration. PubMed: 23469282|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 62 kDa (predicted molecular weight: 55 kDa).|
|IP||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
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.
Immunohistochemistry - Free Floating analysis of mouse brain labelling HDAC1 with ab19845 at 1/100 dilution. HDAC1 was detected in the nuclei (arrows) of dopamine neurons, neuronal marker (magenta) and Hoechst stain (blue).
Brains were post-fixed overnight in phosphate-buffered 4% PFA, and equilibrated in 30% sucrose for 2 days. Brains were sectioned on a cryostat at 30 µm. Sections were stored in a cryoprotective tissue collection solution (25% glycerol, 30% ethylene glycol, 0.05 M phosphate buffer (PB)) at −20°C until use. Immunofluorescence was performed using a free-floating method.
This image is courtesy of an abreview submitted by Prof. Paul Townsend, University of Southampton.
Immunocytochemistry/ Immunofluorescence analysis of African green monkey COS7 cells labeling HDAC1 with ab19845 at 1/200 dilution. Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X100. Cells were blocked in 2% BSA for 1 hour at 25°C, followed by incubation with Anti-HDAC1 antibody (ab19845) in 2% BSA for 1 hour at 25°C. A polyclonal goat anti-rabbit Cy3® secondary antibody was used at 1/250 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.