Anti-HDAC9 antibody [EPR5223]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HDAC9 antibody [EPR5223] (AB109446)

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. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HDAC9 antibody [EPR5223] (AB109446)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - 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).

• IP

Unknown

Immunoprecipitation - Anti-HDAC9 antibody [EPR5223] (AB109446)

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.

All lanes:

Immunoprecipitation - Anti-HDAC9 antibody [EPR5223] (ab109446)

Predicted band size: 111 kDa

false

• WB

Lab

Western blot - Anti-HDAC9 antibody [EPR5223] (AB109446)

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.

All lanes:

Western blot - Anti-HDAC9 antibody [EPR5223] (ab109446)

Predicted band size: 111 kDa

Observed band size: 140 kDa

false

• WB

Unknown

Western blot - Anti-HDAC9 antibody [EPR5223] (AB109446)

All lanes:

Western blot - Anti-HDAC9 antibody [EPR5223] (ab109446) at 1/1000 dilution

Lane 1:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 111 kDa

false