重组Anti-HDAC2抗体[Y461] (ab32117)

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 - ab32117 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32117 was shown to specifically react with HDAC2 when HDAC2 knockout samples were used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  ab32117 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 and 1/10000 respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

ab32117 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 ab32117 at 1/250 dilution 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).

ab32117 staining HDAC2 in MCF-7 (human breast carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control 1: PBS only.

All lanes : Anti-HDAC2 antibody [Y461] (ab32117) at 1/1000 dilution

Lane 1 : Rat brain tissue homogenate at 20 µg
Lane 2 : Rat brain tissue homogenate at 40 µg
Lane 3 : Rat brain tissue homogenate, P1 nuclear fraction at 20 µg
Lane 4 : Rat brain tissue homogenate, P1 nuclear fraction at 40 µg
Lane 5 : Rat brain tissue homogenate, P2 non-nuclear fraction at 20 µg
Lane 6 : Rat brain tissue homogenate, P2 non-nuclear fraction at 40 µg

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/1000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?
Additional bands at: 35 kDa (possible non-specific binding)


Exposure time: 5 minutes

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Anti-HDAC2 antibody [Y461] (ab32117) at 1/2000 dilution + K562 cell lysate

Predicted band size: 55 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

HDAC2 was immunoprecipitated from 1mg of Hela(Human epithelial cell line from cervix adenocarcinoma)whole cell lysate with ab32117at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab32117 at 1/1000 dilution.

Anti-Rabbit IgG (HRP),specific to the non-reduced form of IgG, was used as secondary antibody at 1/1000 dilution.

Lane 1: Hela whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Immunohistochemical analysis of HDAC2 expression in paraffin embedded human breast carcinoma tissue section, using 1/250 ab32117.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with ab32117 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32117, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.