Revision 1

#7556Store 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:

#Q14164

Entrez-Gene Id:

9641

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 IKKepsilon (Met1-Val716) (GenBank Accession No. NM_014002) 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 IKKepsilon kinase. It includes active IKKepsilon 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-PAK1 (Ser144)/PAK2 (Ser141) peptide: 1,775 Daltons. GST-IKKe Kinase: 110 kDa.

Background

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins (1-3). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).
Recently, two homologs of IKKalpha and IKKbeta have been described, called IKKepsilon (also known as IKK-i) and TBK-1 (also known as T2K or NAK), and activation of either of these kinases results in NFkappaB activation. The kinase domain of IKKepsilon is located in its amino-terminus, which shares 30% sequence homology with both IKKalpha and IKKbeta. IKKepsilon is expressed predominantly in immune cells, and may play a special role in the immune response (14-18).

  1. Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.
  2. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
  3. Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
  4. Brown, K. et al. (1995) Science 267, 1485-8.
  5. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  8. Zandi, E. et al. (1997) Cell 91, 243-52.
  9. Karin, M. (1999) Oncogene 18, 6867-74.
  10. DiDonato, J.A. et al. (1997) Nature 388, 548-54.
  11. Mercurio, F. et al. (1997) Science 278, 860-6.
  12. Johnson, L.N. et al. (1996) Cell 85, 149-58.
  13. Delhase, M. et al. (1999) Science 284, 309-13.
  14. Shimada, T. et al. (1999) Int Immunol 11, 1357-62.
  15. Peters, R.T. et al. (2000) Mol Cell 5, 513-22.
  16. Tojima, Y. et al. (2000) Nature 404, 778-82.
  17. Bonnard, M. et al. (2000) EMBO J 19, 4976-85.
  18. Peters, R.T. and Maniatis, T. (2001) Biochim Biophys Acta 1471, M57-62.

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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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
#7556

HTScan® IKKε Kinase Assay Kit

HTScan® IKKε Kinase Assay Kit: Image 1 Expand Image
Figure 3. Dose dependence curve of IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of substrate peptide (#1134) by IKKepsilon kinase. In a 50 µl reaction, increasing amounts of IKKepsilon 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® IKKε Kinase Assay Kit: Image 2 Expand Image
Figure 5. Staurosporine inhibition of IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of IKKepsilon substrate peptide #1134 by IKKepsilon kinase. In a 50 µl reaction, 50 ng IKKepsilon, 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® IKKε Kinase Assay Kit: Image 3 Expand Image
Figure 4. Peptide concentration dependence of IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of substrate peptide (#1134) by IKKepsilon kinase. In a 50 µl reaction, 50 ng of IKKepsilon 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® IKKε Kinase Assay Kit: Image 4 Expand Image
Figure 2. Time course of IKKepsilon kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of IKKepsilon substrate peptide #1134 by IKKepsilon kinase. In a 50 µl reaction, 50 ng IKKepsilon and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® IKKε Kinase Assay Kit: Image 5 Expand Image
Figure 1. IKKepsilon 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: Casein, 10 µg/50 µl, and recombinant IKKepsilon: 100 ng/50 µl.
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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.