Anti-JNK2 抗体 [EP1595Y] (ab76125)

Lanes 1-3: Merged signal (red and green). Green - ab76125 observed at 48 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab76125 Anti-JNK2 antibody [EP1595Y]  was shown to specifically react with JNK2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266355 (knockout cell lysate ab257527) was used.  Wild-type and JNK2 knockout samples were subjected to SDS-PAGE.  ab76125 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

ab76125 at 1/100 dilution staining JNK2 in human breast carcinoma by Immunohistochemistry, Paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: JNK2 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: MCF7 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab76125 observed at 54 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76125 was shown to specifically react with JNK2 when JNK2 knockout samples were used. Wild-type and JNK2 knockout samples were subjected to SDS-PAGE. ab76125 and ab8245 (loading control to GAPDH) were diluted 1/2500 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

JNK2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit monoclonal to JNK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab76125.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 48kDa; JNK2

Overlay histogram showing HeLa cells stained with ab76125 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76125, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.