All lanes : Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] (ab124956) at 1/1000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) Whole cell lysates with 5% NFDM/TBST
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 20J/m2 UV-C then recovery for 1 hour whole cell lysates with 5% NFDM/TBST
Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 20J/m2 UV-C then recovery for 1 hour whole cell lysates. Then the membrane was incubated with alkaline phosphatase with 5% NFDM/TBST
Lane 4 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 20J/m2 UV-C then recovery for 1 hour whole cell lysates. Then the membrane was incubated with lambda phosphatase with 5% NFDM/TBST

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Observed band size: 46,54 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

ab124956, at 1/100 dilution staining JNK1+JNK2+JNK3 in paraffin-embedded Human brain tissue, by Immunohistochemistry.

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

Immunocytochemistry/Immunofluorescence analysis of untreated, Anisomycin treated and Anisomycin + LP treated NIH/3T3 cells labelling JNK1 + JNK2 + JNK3 (phospho T183 + T183 + T221) with ab124956 at a dilution of 1/100 (left) and JNK1 + JNK2 + JNK3 with ab179461 at a dilution of 1/250 (right).

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

The image shows increased nuclear staining after Anisomycin (250ng/ml, 30min) treatment on NIH3T3 cells. The LP treatment decreased the increased nuclear staining caused by Anisomycin.

ab179461 was used as a Pan control for ab124956. The results showed cytoplasmic staining on untreated, Anisomycin and Anisomycin + LP treated NIH3T3 cells.

Overlay histogram showing HeLa cells stained with ab124956 (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 (ab124956, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was goat anti-rabbit Alexa Fluor^® 488 IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Purified ab124956 at 1/70 dilution (2µg) immunoprecipitating JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) in HeLa treated with 25ug/mL anisomycin for 30min whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) treated with 25ug/mL anisomycin for 30min whole cell lysate 10µg
Lane 2 (+): ab124956 + HeLa treated with 25ug/mL anisomycin for 30min whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124956 in HeLa treated with 25ug/mL anisomycin for 30min whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 46, 54 kDa

All lanes : Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] (ab124956) at 1/1000 dilution

Lane 1 : NIH 3T3 cell lysate, untreated
Lane 2 : NIH 3T3 cell lysate, treated with Anisomycin

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Secondary antibody - goat anti-rabbit HRP (ab6721)

Dot blot analysis of JNK1/2/3 (pT183 + pT183 + pT221) peptide (Lane 1) and JNK1/2/3 non-phospho peptide (Lane 2) labelling JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) with ab124956 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Equilibrium disassociation constant (K_D)
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