Interested in promotions? | Click here >>
7549
HTScan® IKKβ Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® IKKβ Kinase Assay Kit #7549

Citations (0)
HTScan® IKKβ Kinase Assay Kit: Image 1

Figure 3. Dose dependence curve of IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, increasing amounts of IKKbeta 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® IKKβ Kinase Assay Kit: Image 2

Figure 5. Staurosporine inhibition of IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of IKKbeta substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng IKKbeta, 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® IKKβ Kinase Assay Kit: Image 3

Figure 4. Peptide concentration dependence of IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng of IKKbeta 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® IKKβ Kinase Assay Kit: Image 4

Figure 2. Time course of IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of IKKbeta substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng IKKbeta and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® IKKβ Kinase Assay Kit: Image 5

Figure 1. IKKbeta 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, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Rb CTF 1.5 µg/50 µl, recombinant IKKbeta: 50 ng/50 µl.

To Purchase # 7549

Product Description

The kit provides a means of performing kinase activity assays with recombinant human IKKbeta kinase. It includes active IKKbeta kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Formula

Peptide substrate, Biotin-peptide: 2,085 Daltons. GST-IKKbeta Kinase: 87 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).

  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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.