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    nek9-antibody-ep7361-ab138488.pdf

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Cell Biology Cell Cycle Cell Division Spindle
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-NEK9 antibody [EP7361] (ab138488)

  • Datasheet
  • SDS
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Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
  • Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)
  • Flow Cytometry (Intracellular) - Anti-NEK9 antibody [EP7361] (ab138488)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)
  • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
  • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)
  • Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)
  • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
  • Anti-NEK9 antibody [EP7361] (ab138488)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP7361] to NEK9
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free

You may also be interested in

Protein
Product image
Recombinant human NEK9 protein (ab125614)
Secondary
Product image
Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
Knockout
Product image
Human NEK9 knockout A-431 cell line (ab270474)

View more associated products

Overview

  • Product name

    Anti-NEK9 antibody [EP7361]
    See all NEK9 primary antibodies
  • Description

    Rabbit monoclonal [EP7361] to NEK9
  • Host species

    Rabbit
  • Tested applications

    Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human NEK9 (N terminal). The exact sequence is proprietary.

  • Positive control

    • WB: A431, NIH3T3, HepG2, HeLa, and THP-1 cell lysates; Rat spleen tissue lysate. IHC-P: Human kidney tissue. ICC: HeLa cells. Flow Cyt (intra): HeLa and NIH/3T3 cells.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. Stable for 12 months at -20°C.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol (glycerin, glycerine), 0.05% BSA, 59% PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP7361
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Cell Division
    • Spindle
    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Other
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases

Associated products

  • Alternative Versions

    • Anti-NEK9 antibody [EP7361] - BSA and Azide free (ab248844)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human NEK9 knockout A-431 cell line (ab270474)
  • KO cell lysates

    • Human NEK9 knockout A-431 cell lysate (ab270497)
  • Positive Controls

    • Hep G2 nuclear extract lysate (ab14660)
    • NIH/3T3 nuclear extract lysate (ab14874)
    • THP-1 nuclear extract lysate (ab14900)
    • HeLa whole cell lysate (ab29545)
    • NIH/3T3 whole cell lysate (ab7179)
    • THP-1 whole cell lysate (ab7913)
  • Recombinant Protein

    • Recombinant human NEK9 protein (ab125614)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab138488 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
Flow Cyt (Intra)
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/1000 - 1/10000. Predicted molecular weight: 107 kDa.
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.

See IHC antigen retrieval protocols.

ICC/IF
1/100.

For unpurified use at 1/250 - 1/500.

Notes
Flow Cyt (Intra)
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/1000 - 1/10000. Predicted molecular weight: 107 kDa.
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.

See IHC antigen retrieval protocols.

ICC/IF
1/100.

For unpurified use at 1/250 - 1/500.

Application notes
Is unsuitable for IP.

Target

  • Function

    Pleiotropic regulator of mitotic progression, participating in the control of spindle dynamics and chromosome separation. Phosphorylates different histones, myelin basic protein, beta-casein, and BICD2. Phosphorylates histone H3 on serine and threonine residues and beta-casein on serine residues. Important for G1/S transition and S phase progression.
  • Tissue specificity

    Most abundant in heart, liver, kidney and testis. Also expressed in smooth muscle cells and fibroblasts.
  • Sequence similarities

    Belongs to the protein kinase superfamily. NEK Ser/Thr protein kinase family. NIMA subfamily.
    Contains 1 protein kinase domain.
    Contains 6 RCC1 repeats.
  • Developmental stage

    Expression varied mildly across the cell cycle, with highest expression observed in G1 and stationary-phase cells.
  • Domain

    Dimerizes through its coiled-coil domain.
  • Post-translational
    modifications

    Autophosphorylated on serine and threonine residues. When complexed with FACT, exhibits markedly elevated phosphorylation on Thr-210. During mitosis, not phosphorylated on Thr-210. Phosphorylated by CDK1 in vitro.
  • Cellular localization

    Cytoplasm. Nucleus.
  • Target information above from: UniProt accession Q8TD19 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 91754 Human
    • Entrez Gene: 217718 Mouse
    • Entrez Gene: 299204 Rat
    • Omim: 609798 Human
    • SwissProt: Q8TD19 Human
    • SwissProt: Q8K1R7 Mouse
    • Unigene: 7200 Human
    • Unigene: 29071 Mouse
    • Alternative names

      • KIAA1995 antibody
      • APUG antibody
      • DKFZp434D0935 antibody
      • LCCS10 antibody
      • MGC138306 antibody
      • MGC16714 antibody
      • NC antibody
      • NEK 9 antibody
      • Nek8 antibody
      • nek9 antibody
      • NEK9_HUMAN antibody
      • Nercc 1 kinase antibody
      • NERCC antibody
      • NERCC1 antibody
      • Nercc1 kinase antibody
      • Never in mitosis A related kinase 9 antibody
      • Never in mitosis A-related kinase 9 antibody
      • NIMA (never in mitosis gene a) related kinase 9 antibody
      • NIMA related kinase 8 antibody
      • NIMA related kinase 9 antibody
      • NIMA related kinase Nek8 antibody
      • NimA related protein kinase 9 antibody
      • NIMA-related kinase 8 antibody
      • NimA-related protein kinase 9 antibody
      • Serine/threonine protein kinase Nek9 antibody
      • Serine/threonine-protein kinase Nek9 antibody
      see all

    Images

    • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution

      Lane 1 : Wild-type A431 cell lysate
      Lane 2 : NEK9 knockout A431 cell lysate
      Lane 3 : HepG2 cell lysate
      Lane 4 : MCF7 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 107 kDa
      Observed band size: 120 kDa why is the actual band size different from the predicted?



      Lanes 1 - 4: Merged signal (red and green). Green - ab138488 observed at 120 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

      ab138488 was shown to react with NEK9 in wild-type A431 cells in western blot. Loss of signal was observed when NEK9 knockout sample was used. Wild-type and NEK9 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab138488 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)
      Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)

      Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling NEK9 with Purified ab138488 at 1:100 dilution (3.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

    • Flow Cytometry (Intracellular) - Anti-NEK9 antibody [EP7361] (ab138488)
      Flow Cytometry (Intracellular) - Anti-NEK9 antibody [EP7361] (ab138488)

      Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue). 

       

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling NEK9 with Purified ab138488 at 1:100 dilution (3.84 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

    • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      Anti-NEK9 antibody [EP7361] (ab138488) at 1000 cells (purified) + Rat spleen lysates at 15 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 107 kDa
      Observed band size: 120 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer: 5% NFDM/TBST

    • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/5000 dilution (purified)

      Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
      Lane 2 : THP-1 (Human monocytic leukemia monocyte) whole cell lysates

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 107 kDa
      Observed band size: 120 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer: 5% NFDM/TBST

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)

      Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling NEK9 with unpurified ab138488 at 1/50 dilution.

    • Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)
      Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)

      Immunofluorescent analysis of HeLa cells labelling NEK9 with unpurified ab138488 at 1/250 dilution.

    • Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      Western blot - Anti-NEK9 antibody [EP7361] (ab138488)
      All lanes : Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution (unpurified)

      Lane 1 : NIH3T3 cell lysate
      Lane 2 : HepG2 cell lysate
      Lane 3 : HeLa cell lysate
      Lane 4 : THP1 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 107 kDa

    • Anti-NEK9 antibody [EP7361] (ab138488)
      Anti-NEK9 antibody [EP7361] (ab138488)

    Protocols

    • Flow cytometry protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (8)

    Publishing research using ab138488? Please let us know so that we can cite the reference in this datasheet.

    ab138488 has been referenced in 8 publications.

    • Xu Z  et al. Decreased Nek9 expression correlates with aggressive behaviour and predicts unfavourable prognosis in breast cancer. Pathology 52:329-335 (2020). PubMed: 32098687
    • O'Regan L  et al. EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7. J Cell Sci 133:N/A (2020). PubMed: 32184261
    • Shrestha BK  et al. NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1. J Biol Chem 295:1240-1260 (2020). PubMed: 31857374
    • Freixo F  et al. NEK7 regulates dendrite morphogenesis in neurons via Eg5-dependent microtubule stabilization. Nat Commun 9:2330 (2018). PubMed: 29899413
    • Levinsohn JL  et al. Somatic Mutations in NEK9 Cause Nevus Comedonicus. Am J Hum Genet 98:1030-1037 (2016). PubMed: 27153399
    • He Y  et al. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530:354-7 (2016). PubMed: 26814970
    • Jung R  et al. The Dual Nature of Nek9 in Adenovirus Replication. J Virol 90:1931-43 (2015). PubMed: 26676776
    • Kurioka D  et al. NEK9-dependent proliferation of cancer cells lacking functional p53. Sci Rep 4:6111 (2014). IHC-P ; Human . PubMed: 25131192

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