Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling JNK1+JNK2+JNK3 with ab179461 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing both cytoplasmic and nuclear staining on HeLa cells. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:-
-ve control 1: - ab179461 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR16797-211] (ab179461) at 1/20000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

 

JNKs originate from three genes that yield ten isoforms through alternative mRNA splicing, including JNK1α1, JNK1β1, JNK2α1, JNK2β1 and JNK3α1, which represent the p46 isoforms, and JNK1α2, JNK1β2, JNK2α2, JNK2β2 and JNK3β2, which represent the p54 isoforms.

Formaldehyde-fixed, NP40 permeabilized Mouse Vascular smooth muscle cells stained for JNK1+JNK2+JNK3 (Green) using ab179461 at 1/200 dilution followed by a Donkey anti-rabbit Alex Fluor® 488 antibody at 1/500 dilution. The nuclear counterstain was DAPI (Blue). 

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling JNK1+JNK2+JNK3 with purified ab179461 at 1/180 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR16797-211] (ab179461) at 1/5000 dilution

Lane 1 : Neuro-2a (Mouse neuroblastoma cells) whole cell lysates
Lane 2 : UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates
Lane 3 : MDCK (Canine kidney cell line) whole cell lysates
Lane 4 : MDBK (Bovine kidney cell line) whole cell lysates
Lane 5 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/dilution buffer: 5% NFDM/TBST.

 

JNKs originate from three genes that yield ten isoforms through alternative mRNA splicing, including JNK1α1, JNK1β1, JNK2α1, JNK2β1 and JNK3α1, which represent the p46 isoforms, and JNK1α2, JNK1β2, JNK2α2, JNK2β2 and JNK3β2, which represent the p54 isoforms.

JNK1+JNK2+JNK3 were immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell extract with ab179461 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab179461 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: Jurkat whole cell extract. Lane 2: PBS instead of Jurkat whole cell extract.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

 

JNKs originate from three genes that yield ten isoforms through alternative mRNA splicing, including JNK1α1, JNK1β1, JNK2α1, JNK2β1 and JNK3α1, which represent the p46 isoforms, and JNK1α2, JNK1β2, JNK2α2, JNK2β2 and JNK3β2, which represent the p54 isoforms.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR16797-211] (ab179461) at 1/20000 dilution

Lane 1 : Human JNK3 full length recombinant protein containing a proprietary tag.
Lane 2 : Human JNK2 full length recombinant protein containing a proprietary tag.
Lane 3 : Human JNK1 full length recombinant protein containing a His tag.

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa
Additional bands at: 48 kDa (possible tagged protein), 71 kDa (possible tagged protein), 71 kDa (possible tagged protein)

Human JNK1, JNK2 and JNK3 full length recombinant proteins are from commercial sources. JNK1 and JNK2 have a proprietary tag, JNK3 has a His tag.

 

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR16797-211] (ab179461) at 1/1000 dilution

Lane 1 : Zebrafish lysate
Lane 2 : X. tropicalis lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/dilution buffer: 5% NFDM/TBST.

 

Zebrafish has only one JNK isoform, JNK1 with a MW of 44kDa. So there is only one band in Zebrafish.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR16797-211] (ab179461) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : Rat heart lysate
Lane 4 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma) lysate
Lane 6 : NIH/3T3 (Mouse embyro fibroblast cells) lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 46,54 kDa why is the actual band size different from the predicted?

Blocking/dilution buffer: 5% NFDM/TBST.

 

JNKs originate from three genes that yield ten isoforms through alternative mRNA splicing, including JNK1α1, JNK1β1, JNK2α1, JNK2β1 and JNK3α1, which represent the p46 isoforms, and JNK1α2, JNK1β2, JNK2α2, JNK2β2 and JNK3β2, which represent the p54 isoforms.