Anti-JNK2 抗体 [EP1595Y] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

This IHC data was generated using the same anti-JNK2 antibody clone, EP1595Y, in a different buffer formulation (cat# 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76125).

• IP

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Immunoprecipitation - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76125).

All lanes:

Immunoprecipitation - Anti-JNK2 antibody [EP1595Y] (<a href='/products/primary-antibodies/jnk2-antibody-ep1595y-ab76125'>ab76125</a>)

Predicted band size: 48 kDa

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Exposure time: 20min

• ELISA

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ELISA - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

This data was developed using ab76125, the same antibody clone in a different buffer formulation.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.

• WB

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Western blot - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

This WB data was generated using the same anti-JNK2 antibody clone, EP1595Y, in a different buffer formulation (cat# 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, 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.

All lanes:

Western blot - Anti-JNK2 antibody [EP1595Y] (<a href='/products/primary-antibodies/jnk2-antibody-ep1595y-ab76125'>ab76125</a>)

Predicted band size: 48 kDa

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• WB

Lab

Western blot - Anti-JNK2 antibody [EP1595Y] - BSA and Azide free (AB227986)

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

All lanes:

Western blot - Anti-JNK2 antibody [EP1595Y] (<a href='/products/primary-antibodies/jnk2-antibody-ep1595y-ab76125'>ab76125</a>) 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 (<a href='/products/cell-lines/human-mapk9-jnk2-knockout-hek-293t-cell-line-ab266355'>ab266355</a>)

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 (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 48 kDa

Observed band size: 48 kDa

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