Recombinant Anti-JAK2 antibody [EPR108(2)] (ab108596)

Lanes 1- 4: Merged signal (red and green). Green - ab108596 observed at 131 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab108596 was shown to react with JAK2 in Wild-Type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267113 (knockout cell lysate ab256963) was used. Wild-Type A549 and JAK2 knockout A549 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab108596 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling JAK2 with purified ab108596 at 1/150 dilution (8.5μg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, a goat anti-rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1/1000 dilution. Ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used as the counter stain at 1/200 (2.5 μg/ml). PBS instead of the primary antibody was the negative control. DAPI was used as a nuclear counterstain.

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

Immunocytochemistry/Immunofluorescence analysis of Jurkat (Human T cell leukemia cells from peripheral blood) cells labelling JAK2 with unpurified ab108596 at 1/300 (7.0 μg/mL). Cells were fixed with 4% Paraformaldehydeand permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/mL) was used as the secondary antibody. Nuclei were stained with DAPI (blue).

Confocal image showing nuclear and cytoplasmic staining on Jurkat cell line. 

Immunocytochemistry/Immunofluorescence analysis of Ramos (Human Burkitt's lymphoma) cells labelling JAK2 with unpurified 108596 at 1/300 (7.0 μg/mL). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/mL) was used as the secondary antibody. Nuclei were stained with DAPI (blue).

Confocal image showing nuclear and cytoplasmic staining on Ramos cell line.

ab108596 at 1/80 dilution (20 µg/mL) immunoprecipitating JAK2 in K562 (Human chronic myelogenous leukemia lymphoblast)  cell lysate.

Lane 1 (input): K562(Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10µg

Lane 2 (+): K562 whole cell lysate, 350µg  + ab108596, 2µg

Lane 3 (-): K562 cell lysate, 350µg  + rabbit IgG (ab172730), 2µg

For western blotting, ab108596 at 1/500 dilution (3.0 µg/mL) and ab131366 VeriBlot for IP (HRP) was used at 1/1000.

Blocking and dilution buffer: 5% NFDM/TBST.