Lane 1: Wild type HAP1 whole cell lysate (40 µg)
Lane 2: KDM3A knockout  HAP1 whole cell lysate (40 µg)
Lane 3: HeLa whole cell lysate (40 µg)
Lane 4: Hek293 whole cell lysate (40 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab191389 observed at 150 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab191389 was shown to specifically react with KDM3A when KDM3A knockout samples were used. Wild-type and KDM3A knockout samples were subjected to SDS-PAGE.  Ab191389 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 500 dilution and 1/10000 dilution 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.

 

All lanes : Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389) at 1/1000 dilution

Lane 1 : Untreated HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) treated with 100 µM CoCl2 for 24 hours whole cell lysate
Lane 3 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 100 µM CoCl2 for 24 hours whole cell lysate
Lane 5 : Untreated Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 6 : Jurkat (Human T cell leukemia cell line from peripheral blood) treated with 100 µM CoCl2 for 24 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 100000 mg/ml

Predicted band size: 147 kDa
Observed band size: 100,147,75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 15 seconds; Lane 3 and 4: 30 seconds; Lane 5 and 6: 5 seconds.

 

Anti-KDM3A / JHDM2A antibody [EPR18875] (ab191389) at 1/1000 dilution + Ramos (Human Burkitt's lymphoma cell line) whole cell lysate

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 147 kDa
Observed band size: 147 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on spermatogenic cells of human testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Weak nuclear staining on cancer cells of human bladder cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling KDM3A / JHDM2A with ab191389 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Weak nuclear staining on cancer cells of human lung cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

KDM3A / JHDM2A was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab191389 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab191389 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Ramos whole cell lysate 10µg (Input).

Lane 2: ab191389 IP in Ramos whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab191389 in Ramos whole cell lysate.

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

Exposure time: 10 seconds.