All lanes : Anti-JAK3 antibody [EP909Y] (ab45141) at 1/1000 dilution

Lane 1 : TF-1 (Human Erythroleukemia erythroblast) whole cell lysate
Lane 2 : HaCaT (Human skin keratinocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 125 kDa
Observed band size: 125 kDa


Exposure time: 3 minutes

Blocking/Diluting buffer and concentration 5% NFDM/TBST

The expression profile observed in HaCaT is consistent with the literature (PMID: 11709700).
Negative control: HaCaT (PMID: 11709700)

Immunocytochemistry/Immunofluorescence analysis of KARPAS-299 (human anaplastic large cell lymphoma) labelling JAK3 with ab45141 at a dilution of 1:100 dilution (12 µg/ml). Cells were fixed with 100% Methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) (1:1000 dilution (2 µg/ml)) was used as the secondary antibody. The cells were co-stained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

All lanes : Anti-JAK3 antibody [EP909Y] (ab45141) at 1.21 µg/ml

Lane 1 : KARPAS-299 (Human anaplastic large cell lymphoma ) whole cell lysate
Lane 2 : HEL (Human Erythroleukemia erythroblast ) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 125 kDa
Observed band size: 125 kDa


Exposure time: 26 seconds

Blocking and diluting buffer: 5% NFDM/TBST.

Lane 1 (input): TF-1 (Human Erythroleukemia erythroblast) whole cell lysate, 10µg
Lane 2 (+): TF-1 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab45141 in TF-1 whole cell lysate.

ab45141 at 1/60 immunoprecipitating JAK3 in TF-1 whole cell lysate. For western blotting, ab45141 was used as a primary antibody at 1/500 dilution (2.42 µg/ml). ab131366, VeriBlot for IP Detection Reagent was used for detection at 1/1000 dilution. Blocking and diluting buffer used was 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections) analysis of Human NK cell lymphoma tissue sections labeling JAK3 using purified ab45141. Samples were incubated the primary antibody at 1:2000 dilution (0.60 μg/ml). Hematoxylin was used as a counterstain. PBS instead of primary antibody was used for negative control. A ready to use ImmunoHistoProbe one step HRP Polymer at 1:0 dilution was used as the secondary antibody. Heatm mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections) analysis of Human large B cell lymphoma tissue sections labeling JAK3 using purified ab45141. Samples were incubated the primary antibody at 1:2000 dilution (0.60 μg/ml). Hematoxylin was used as a counterstain. PBS instead of primary anitbody was used for negative control. A ready to use ImmunoHistoProbe one step HRP Polymer at 1:0 dilution was used as the secondary antibody. Heatm mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Anti-JAK3 antibody [EP909Y] (ab45141) at 1/2000 dilution + TF-1 (Human bone marrow erythroleukemia cell line) cell lysate

Predicted band size: 125 kDa
Observed band size: 125 kDa

Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with ab45141 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab45141, 1/100 dilution) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was rabbit IgG (1 µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.