Recombinant Anti-ZIP Kinase antibody [EPR18809-86] (ab210528)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: ZIP Kinase knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab210528 observed at 53 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab210528 was shown to specifically react with ZIP Kinase in wild type cells as signal was lost in ZIP Kinase knockout cells. Wild-type and ZIP Kinase knockout samples were subjected to SDS-PAGE. Ab210528 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1 : Human ZIP Kinase fragment recombinant protein
Lane 2 : Human DAP Kinase 2 fragment recombinant protein
Lane 3 : Human DAP Kinase 1 fragment 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: 53 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Human ZIP Kinase fragment recombinant protein contain aa13-275 with a His-Tag®. Human DAP Kinase 2 fragment recombinant protein contain aa23-285 with a His-Tag®. Human DAP Kinase 1 fragment recombinant protein contain aa13-275 with a His-Tag®. All three recombinant human fragment proteins were made in-house.

The antibody reacts weakly with DAP kinase 1 and DAP kinase 2; however bands of their appropriate MW (159 kD, 42 kD) are not detected in the tissue lysates.

All lanes : Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human bladder 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: 53 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 20124481).

All lanes : Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : C2C12 (Mouse myoblast cell line) whole cell lysate
Lane 5 : L6 (Rat skeletal muscle cell line) whole cell lysate
Lane 6 : Mouse bladder lysate
Lane 7 : Rat bladder lysate
Lane 8 : A549 (Human lung carcinoma 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: 53 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1, 2, 3, 4, 5, 6 and 7: 30 seconds; Lane 8: 5 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 20124481).

All lanes : Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate at 10 µg
Lane 2 : Mouse kidney tissue lysate at 10 µg
Lane 3 : Mouse spleen tissue lysate at 10 µg
Lane 4 : Rat brain tissue lysate at 10 µg
Lane 5 : Rat heart tissue lysate at 10 µg
Lane 6 : Rat kidney tissue lysate at 10 µg
Lane 7 : Rat spleen tissue lysate at 10 µg
Lane 8 : C6 (Rat glial tumor cell line) whole cell lysate at 20 µg
Lane 9 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg
Lane 10 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg
Lane 11 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

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

Predicted band size: 53 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1, 2 and 3: 30 seconds; Lane 4, 5, 6 and 7: 3 minutes; Lane 8, 9, 10 and 11: 30 seconds.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 20124481)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ZIP Kinase with ab210528 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm and nuclear staining on HeLa cell line. The nuclear counter stain 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: ab210528 at 1/100 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) cells labeling ZIP Kinase with ab210528 at 1/100 dilution, followed by Goat anti-rabbit IgG (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 counter stain 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: ab210528 at 1/100 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed C2C12 (Mouse myoblast cell line) cells labeling ZIP Kinase with ab210528 at 1/120 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. 

 

 

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling ZIP Kinase with ab210528 at 1/600 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. 

Note:Cells were permeabilised with 90% methanol -1XPBS (-20?, 30min).