All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR18841-95] (ab208035) at 1/2000 dilution

Lane 1 : Human JNK1 full length recombinant protein
Lane 2 : Human JNK2 full length recombinant protein
Lane 3 : Human JNK3 full length recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48, 53 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 & 2: 5 seconds; Lane 3: 15 seconds.

Human JNK1 full length recombinant protein contains aa1-427 with a His-Tag®. Human JNK2 full length recombinant protein contains aa1-424 with a His-Tag®. Human JNK3 full length recombinant protein contains aa1-464 with a His-Tag®.

All three recombinant human full length proteins were made in-house.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR18841-95] (ab208035) at 1/2000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 48, 53 kDa
Observed band size: 46-54 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR18841-95] (ab208035) at 1/2000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48, 53 kDa
Observed band size: 46-54 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR18841-95] (ab208035) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat kidney lysate
Lane 7 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48, 53 kDa
Observed band size: 46-54 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 4 seconds; Lane 2,3 & 4: 30 seconds; Lane 5: 15 seconds; Lane 6 & 7: 3 minutes.

All lanes : Anti-JNK1+JNK2+JNK3 antibody [EPR18841-95] (ab208035) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48, 53 kDa
Observed band size: 46-54 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling JNK1+JNK2+JNK3 with ab208035 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasm and weak nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab208035 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cell line labeling JNK1+JNK2+JNK3 with ab208035 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasm and nuclear staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab208035 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cell line labeling JNK1+JNK2+JNK3 with ab208035 at 1/100 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat anti-Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling JNK1+JNK2+JNK3 with ab208035 at 1/100 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat anti-Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

JNK1+JNK2+JNK3 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208035 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab208035 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate, 10ug (Input).

Lane 2: ab208035 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab208035 in HeLa whole cell lysate.

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

Exposure time: 10 seconds.