Revision 1

#7555Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P51955

Entrez-Gene Id:

4751

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol. Store at -80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human NEK2 (Met1-Arg445) (GenBank Accession No. NM_002497) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing kinase activity assays with recombinant human NEK2 kinase. It includes full-length human active NEK2 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Formula Peptide substrate, Biotin-CREB (Ser133) peptide: 2,326 Daltons. GST-NEK2 Kinase: 83 kDa.

Background

The NEK family of protein kinases is composed of 11 members in humans that share an amino-terminal catalytic domain related to NIMA, a serine/threonine kinase identified in Aspergillus nidulans. While NIMA is critical for cell cycle progression in fungus, the function of NEK kinases in mammalian cells is largely unknown. NEK1 was first identified by screening mouse cDNA expression libraries and was demonstrated to have dual specificity kinase activity on both tyrosine and serine/threonine sites (1). NEK2 most closely resembles fungal NIMA in its primary structure and is believed to promote the splitting of duplicated centrosomes at the onset of mitosis (2,3). NEK3 is predominantly a cytoplasmic enzyme and its activity shows marginal variation throughout the cell cycle (4). NEK4 is ubiquitously expressed and its expression and subcellular location are not associated with cell cycle (5). NEK6/7 have been suggested to phosphorylate and activate p70 S6 kinase in vitro (6). Expression of an inactive NEK6 mutant arrests cells in M phase and interferes with chromosome segregation (7). NEK8 activity is not cell cycle regulated and may play a role in cell cycle independent microtubule dynamics (8). NEK9 is activated during mitosis and may participate in the activation of NEK6/7 during mitosis (9,10).

  1. Letwin, K. et al. (1992) EMBO J. 11, 3521-3531.
  2. Fry, A.M. et al. (1995) J. Biol. Chem. 270, 12899-12905.
  3. Fry, A.M. (2002) Oncogene 21, 6184-6194.
  4. Tanaka, K. and Nigg, E.A. (1999) J. Biol. Chem. 274, 13491-13497.
  5. Hayashi, K. et al. (1999) Biochem. Biophys. Res. Commun. 264, 449-456.
  6. Belham, C. et al. (2001) Curr. Biol. 11, 1155-1167.
  7. Yin, M.J. et al. (2003) J. Biol. Chem. 278, 52454-52460.
  8. Holland, P.M. et al. (2002) J. Biol. Chem. 277, 16229-16240.
  9. Belham, C. et al. (2003) J. Biol. Chem. 278, 34897-34909.
  10. Roig, J. et al. (2002) Genes Dev. 16, 1640-1658.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
#7555

HTScan® NEK2 Kinase Assay Kit

HTScan® NEK2 Kinase Assay Kit: Image 1 Expand Image
Figure 1. NEK2 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP , 2.5 µg/50 µl PEG20,000, Substrate: Myelin basic protein, 5 µg/50µl, and recombinant NEK2: 100 ng/50 µl.
HTScan® NEK2 Kinase Assay Kit: Image 2 Expand Image
Figure 3. Dose dependence curve of NEK2 kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by NEK2 kinase. In a 50 µl reaction, increasing amounts of NEK2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® NEK2 Kinase Assay Kit: Image 3 Expand Image
Figure 5. Staurosporine inhibition of NEK2 kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of NEK2 substrate peptide (#1331) by NEK2 kinase. In a 50 µl reaction, 50 ng NEK2, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® NEK2 Kinase Assay Kit: Image 4 Expand Image
Figure 4. Peptide concentration dependence of NEK2 kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by NEK2 kinase. In a 50 µl reaction, 50 ng of NEK2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® NEK2 Kinase Assay Kit: Image 5 Expand Image
Figure 2. Time course of NEK2 kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of NEK2 substrate peptide (#1331) by NEK2 kinase. In a 50 µl reaction, 50 ng NEK2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.