Cell Signaling Technology

Product Pathways - HTScan Kinase Assay Kits

HTScan® IKKε Kinase Assay Kit #7556

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Kit Includes Quantity
Phospho-(Ser/Thr) Phe Antibody # 9631 30 microliters
Kinase Buffer (10X) # 9802 15 milliliters
ATP (10 mM) # 9804 1 milliliters
PAK1 (Ser144)/ PAK2 (Ser141) Biotinylated Peptide # 1134 1.25 milliliters
IKKε Kinase # 7553 5 micrograms

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 Weights

Peptide substrate, Biotin-PAK1 (Ser144)/PAK2 (Ser141) peptide: 1,775 Daltons. GST-IKKe Kinase: 110 kDa.

Peptide Core Sequence

YMS*FT

Kinase Assay - Radiometric

Kinase Assay - Radiometric

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.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

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.)

Kinase Assay - DELFIA

Kinase Assay - DELFIA

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.)


Kinase Assay - DELFIA

Kinase Assay - DELFIA

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.)

Kinase Assay - DELFIA

Kinase Assay - DELFIA

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.)

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.

Quality Control

The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-(Ser/Thr) Phe Antibody #9631 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified IKKepsilon kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the IKKepsilon kinase was determined using a radiometric filter binding assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose dependency [Fig.4] assays were performed to verify IKKepsilon activity using the IKKepsilon substrate peptide provided in this kit. IKKepsilon sensitivity to the inhibitor staurosporine was measured using the IKKepsilon substrate peptide provided in this kit [Fig.5].

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. IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends on phosphorylation; Ser177 and Ser181 in the activation loop of IKKβ (176 and 180 in IKKα) are the specific sites whose phosphorylation 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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  5. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
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  14. Shimada , T. et al. (1999) Int. Immunol. 11(8) , 1357-1362.
  15. Peters, R. T. et al. (2000) Mol. Cell 5(3) , 513-522.
  16. Tojima, Y. et al. (2000) Nature 404(6779) , 778-782.
  17. Bonnard, M. et al. (2000) Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription. EMBO J. 19, 4976-4985.
  18. Peters , R.S. and Maniatis, T. (2001) Biochim. Biophys. Acta. 1471(2) , 57-62.

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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