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7549
HTScan® IKKβ Kinase Assay Kit
Cellular Assay Kits

HTScan® IKKβ Kinase Assay Kit #7549

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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.)

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.)

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.)

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.)

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.

Peptide Core Sequence:

DS*GLDS*M

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).

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  2. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
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  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.

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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.