Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat Cerebrum tissue labelling HDAC9 with ab109446 at 1/200 dilution for 20 minutes at room temperature. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Nuclear staining on rat cerebrum. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: HDAC9 (KO) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)
Lane 4: Raji whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109446 observed at 140 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109446 was shown to specifically recognize HDAC9 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when HDAC9 knockout samples were usedexamined. Wild-type and HDAC9 knockout samples were subjected to SDS-PAGE. Ab109446 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse Cerebrum tissue labelling HDAC9 with ab109446 at 1/200 dilution for 20 minutes at room temperature. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Nuclear staining on mouse cerebrum. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

ab109446 at 1/250 dilution staining HDAC9 in Human colon by Immunohistochemistry, Paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab109446 at 1/100 dilution staining HDAC9 in HeLa cells by Immunofluorescence.

HDAC9 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab109446 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using 260035 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 μg

Lane 2: K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109446 in K-562 whole cell lysate

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

Exposure time: 111 seconds.

Flow cytometry of K-562 Human chronic myelogenous leukemia lymphoblast cells staining HDAC9 with ab109446 (red) at 1:250. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used at 1:2000 as a secondary. Rabbit monoclonal IgG (ab172730)  (Black) was used as an isotype control. Cell without incubation with primary antibody and secondary antibody (Blue) used as an unlabelled control. Fixed with 4% paraformaldehyde. Permeabilised with 90% methanol.

All lanes : Anti-HDAC9 antibody [EPR5223] (ab109446) at 1/10000 dilution

Lane 1 : K562 cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : Raji cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 111 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?