Recombinant Anti-HDAC9 antibody [EPR5223] (ab109446)

Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling HDAC9 with Purified ab109446 at 1:100 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling HDAC9 with Purified ab109446 at 1:100 dilution (10 µg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling HDAC9 with Purified ab109446 at 1:1000 dilution (1.10 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

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.

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 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.