Anti-JAK3 antibody [EP909Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JAK3 antibody [EP909Y] (AB45141)

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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-JAK3 antibody [EP909Y] (AB45141)

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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit IgG (1μg/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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-JAK3 antibody [EP909Y] (AB45141)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JAK3 antibody [EP909Y] (AB45141)

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).

• IP

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Immunoprecipitation - Anti-JAK3 antibody [EP909Y] (AB45141)

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.

All lanes:

Immunoprecipitation - Anti-JAK3 antibody [EP909Y] (ab45141)

Predicted band size: 125 kDa

false

• WB

Lab

Western blot - Anti-JAK3 antibody [EP909Y] (AB45141)

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)

All lanes:

Western blot - Anti-JAK3 antibody [EP909Y] (ab45141) at 1/1000 dilution

Lane 1:

TF-1 (Human Erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 2:

HaCaT (Human skin keratinocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 125 kDa

Observed band size: 125 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-JAK3 antibody [EP909Y] (AB45141)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-JAK3 antibody [EP909Y] (ab45141) at 1.21 µg/mL

Lane 1:

KARPAS-299 (Human anaplastic large cell lymphoma ) whole cell lysate at 20 µg

Lane 2:

HEL (Human Erythroleukemia erythroblast ) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 125 kDa

Observed band size: 125 kDa

false

Exposure time: 26s

• WB

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Western blot - Anti-JAK3 antibody [EP909Y] (AB45141)

All lanes:

Western blot - Anti-JAK3 antibody [EP909Y] (ab45141) at 1/2000 dilution

All lanes:

TF-1 (Human bone marrow erythroleukemia cell line) cell lysate

Predicted band size: 125 kDa

Observed band size: 125 kDa

false