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7556
HTScan® IKKε Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® IKKε Kinase Assay Kit #7556

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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.)
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.)
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.)
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.)
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.
Inquiry Info.# 7556

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).

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