Anti-JNK2 antibody [EP1595Y]

7 product images

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  Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125)

  Lanes 1-3: Merged signal (red and green). Green - ab76125 observed at 48 kDa. Red - loading control Anti-GAPDH antibody [6C5] - 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 Human MAPK9 (JNK2) knockout HEK-293T cell line ab266355 (knockout cell lysate Human MAPK9 knockout HEK-293T cell lysate ab257527) was used. Wild-type and JNK2 knockout samples were subjected to SDS-PAGE. ab76125 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.

  All lanes: Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125) at 1/1000 dilution

  Lane 1: Wild-type HEK293T cell lysate at 20 µg

  Lane 2: Western blot - Human MAPK9 (JNK2) knockout HEK-293T cell line (Human MAPK9 (JNK2) knockout HEK-293T cell line ab266355)

  Lane 2: MAPK9 knockout HEK293T cell lysate at 20 µg

  Lane 3: MCF7 cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Predicted band size: 48 kDa

  Observed band size: 48 kDa

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  Immunoprecipitation - Anti-JNK2 antibody [EP1595Y] (ab76125)

  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) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).

  Band: 48kDa; JNK2

  All lanes: Immunoprecipitation - Anti-JNK2 antibody [EP1595Y] (ab76125)

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 48 kDa

  Exposure time: 20min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JNK2 antibody [EP1595Y] (ab76125)

  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.

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  Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125)

  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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125)

  Predicted band size: 48 kDa

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  Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125)

  All lanes: Western blot - Anti-JNK2 antibody [EP1595Y] (ab76125) at 1/50000 dilution

  All lanes: HeLa cell lysate at 10 µg

  Secondary

  All lanes: goat anti-rabbit-HRP at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 48 kDa

  Observed band size: 46 kDa, 54 kDa

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  ELISA - Anti-JNK2 antibody [EP1595Y] (ab76125)

  ELISA analysis of Human JNK2 recombinant protein at 250 ng/mL with ab76125. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

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  Flow Cytometry (Intracellular) - Anti-JNK2 antibody [EP1595Y] (ab76125)

  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) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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.