Recombinant 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
Related conjugates and formulations
Overview
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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.
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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.
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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
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
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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...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP7361 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
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 |
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Flow Cyt (Intra) |
1/10 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB |
1/1000 - 1/10000. Predicted molecular weight: 107 kDa.
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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. |
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ICC/IF |
1/100.
For unpurified use at 1/250 - 1/500. |
Notes |
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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. |
ICC/IF
1/100. For unpurified use at 1/250 - 1/500. |
Target
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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
modificationsAutophosphorylated 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. - Information by UniProt
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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
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Alternative names
- KIAA1995 antibody
- APUG antibody
- DKFZp434D0935 antibody
see all
Images
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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.
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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.
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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).
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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.
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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
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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
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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.
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Immunofluorescent analysis of HeLa cells labelling NEK9 with unpurified ab138488 at 1/250 dilution.
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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
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (8)
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