Anti-IL-2 antibody [EPR20055] (ab207325) at 1/1000 dilution + Human IL2 active recombinant protein at 0.01 µg

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

Predicted band size: 17 kDa
Observed band size: 14 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

This data was developed using ab207325, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Human IL2 active recombinant protein contains aa21-153.

All lanes : Anti-IL-2 antibody [EPR20055] (ab207325) at 1/5000 dilution

Lane 1 : Untreated Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate treated with 40nM Phorbol-12-myristate-13-acetate, 2 µM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 17 kDa
Observed band size: 14 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

This data was developed using ab207325, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with the literature, and the expression level can be induced in activated Jurkat cell (PMID: 10555752).

This data was developed using ab207325, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells, untreated or treated with 40 nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight, labeling IL2 with ab207325 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Jurkat cells. The signal is induced after treatment with 40 nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight. The nuclear counter stain is DAPI (blue). Tubulin is detected with (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red). Secondary antibody only controls on non treated and treated cells: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

This data was developed using ab207325, the same antibody clone in a different buffer formulation.IL2 was immunoprecipitated from 0.35 mg of Jurkat (Human T cell leukemia cell line from peripheral blood) treated with 40nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight lysate with ab207325 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab207325 at 1/200 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. Lane 1: Jurkat treated with 40nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight lysate 10µg (Input). Lane 2: ab207325 IP in Jurkat treated with 40nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight lysate. Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab207325 in Jurkat treated with 40nM Phorbol-12-myristate-13-acetate, 2 μM A23187, and 300 ng/ml Brefeldin A (45 min delay) overnight lysate. Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 3 minutes.