Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124956).

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124956).

• IP

Lab

Immunoprecipitation - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

This data was developed using ab124956, the same antibody clone in a different buffer formulation.
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:

Immunoprecipitation - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] (<a href='/en-us/products/primary-antibodies/jnk1-jnk2-jnk3-phospho-t183t183t221-antibody-epr5693-ab124956'>ab124956</a>)

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124956).

• OI-RD Scanning

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OI-RD Scanning - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• Dot

Lab

Dot Blot - Anti-JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) antibody [EPR5693] - BSA and Azide free (AB219584)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124956).