Anti-ZIP Kinase antibody [EPR18809-86]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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°C, 30min).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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.

• WB

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Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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:

Western blot - 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 at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

C2C12 (Mouse myoblast cell line) whole cell lysate at 20 µg

Lane 5:

L6 (Rat skeletal muscle cell line) whole cell lysate at 20 µg

Lane 6:

Mouse bladder lysate at 20 µg

Lane 7:

Rat bladder lysate at 20 µg

Lane 8:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 53 kDa

Observed band size: 50 kDa

false

• WB

Supplier Data

Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human bladder lysate at 10 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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:

Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (ab210528) at 1/1000 dilution

Lane 1:

Human ZIP Kinase fragment recombinant protein at 0.01 µg

Lane 2:

Human DAP Kinase 2 fragment recombinant protein at 0.01 µg

Lane 3:

Human DAP Kinase 1 fragment recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 53 kDa

Observed band size: 29 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - 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:

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

Predicted band size: 53 kDa

false

• WB

Supplier Data

Western blot - Anti-ZIP Kinase antibody [EPR18809-86] (AB210528)

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)

All lanes:

Western blot - 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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 53 kDa

Observed band size: 50 kDa

false