Validated using a knockout cell line
Recombinant
RabMAb

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

Overview

  • Product name

    Anti-ZIP Kinase antibody [EPR18809-86]
    See all ZIP Kinase primary antibodies
  • Description

    Rabbit monoclonal [EPR18809-86] to ZIP Kinase
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cytmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Human ZIP Kinase aa 1-300. The exact sequence is proprietary.
    Database link: O43293

  • Positive control

    • WB: Human ZIP Kinase fragment recombinant protein; Human fetal liver, fetal heart, fetal kidney and bladder lysates; Jurkat, A431, HeLa, C2C12, L6, A549, C6, RAW 264.7, PC-12 and NIH/3T3 cell lysates; Mouse and rat bladder lysates; Mouse brain, kidney and spleen lysates; Rat brain, heart, kidney and spleen lysates. ICC/IF: HeLa and NIH/3T3 cells. Flow Cyt: C2C12 and NIH/3T3 cells.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab210528 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000. Detects a band of approximately 50 kDa (predicted molecular weight: 53 kDa).
ICC/IF 1/100.
Flow Cyt 1/120.

Target

  • Function

    Serine/threonine kinase which acts as a positive regulator of apoptosis. Phosphorylates histone H3 on 'Thr-11' at centromeres during mitosis. Regulates myosin light chain phosphatase through phosphorylation of MYPT1 thereby regulating the assembly of the actin cytoskeleton, cell migration, invasiveness of tumor cells, smooth muscle contraction and neurite outgrowth. Involved in the formation of promyelocytic leukemia protein nuclear body (PML-NB), one of many subnuclear domains in the eukaryotic cell nucleus, and which is involved in oncogenesis and viral infection.
  • Sequence similarities

    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. DAP kinase subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications

    Ubiquitinated. Ubiquitination mediated by the UBE2D3 E3 ligase does not lead to proteasomal degradation, but influences promyelocytic leukemia protein nuclear bodies (PML-NBs) formation in the nucleus.
    Autophosphorylated. Phosphorylated by ROCK1.
  • Cellular localization

    Nucleus. Cytoplasm. Nucleus > PML body. Relocates to the cytoplasm on binding PAWR where the complex appears to interact with actin filaments (By similarity). Localizes to promyelocytic leukemia protein nuclear bodies (PML-NBs). Associates to centromeres from prophase to anaphase.
  • Information by UniProt
  • Database links

  • Alternative names

    • DAP kinase 3 antibody
    • DAP like kinase antibody
    • DAP-like kinase antibody
    • Dapk 3 antibody
    • DAPK3 antibody
    • DAPK3_HUMAN antibody
    • Death associated kinase 3 antibody
    • Death associated protein kinase 3 antibody
    • Death-associated protein kinase 3 antibody
    • Dlk antibody
    • EC 2.7.11.1 antibody
    • FLJ36473 antibody
    • MYPT1 kinase antibody
    • ZIP antibody
    • ZIP kinase antibody
    • ZIP kinase isoform antibody
    • ZIP-kinase antibody
    • ZIPK antibody
    • zipper-interacting protein kinase antibody
    see all

Images

  • 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 lysate at 10 µg
    Lane 2 : Mouse kidney lysate at 10 µg
    Lane 3 : Mouse spleen lysate at 10 µg
    Lane 4 : Rat brain lysate at 10 µg
    Lane 5 : Rat heart lysate at 10 µg
    Lane 6 : Rat kidney lysate at 10 µg
    Lane 7 : Rat spleen 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.

  • 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.

  • 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).

References

ab210528 has not yet been referenced specifically in any publications.

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