Product Pathways - HTScan Kinase Assay Kits
HTScan® IKKβ Kinase Assay Kit #7549
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-IκBα (Ser32/36) (5A5) Mouse mAb # 9246 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| IKKβ Kinase # 7548 | 5 micrograms |
| IκB-α (Ser32) Biotinylated Peptide # 1146 | 1.25 milliliters |
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 Weights
Peptide substrate, Biotin-peptide: 2,085 Daltons. GST-IKKbeta Kinase: 87 kDa.
Peptide Core Sequence
DS*GLDS*M
Kinase Assay - Radiometric
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.
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human IKKbeta (Met1-Ser756) (GenBank Accession No. AF029684) 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-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified IKKbeta kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the IKKbeta kinase was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify IKKbeta activity using the IKKbeta substrate peptide provided in this kit. IKKbeta sensitivity to the inhibitor staurosporine was measured using the IKKbeta 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).
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Application References
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