Product Pathways - NF-kappaB Signaling
IKKβ Kinase #7548
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.
Description
Purified recombinant full length human IKKbeta kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system from a construct containing a full length human IKKbeta cDNA kinase domain (Met1-Ser756) (GenBank accession No. AF029684) fragment amino-terminally fused to a GST-HIS6-Thrombin cleavage site. The protein was then purified by one-step affinity purification using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-IKKbeta fusion protein was analyzed using SDS/PAGE followed by anti-IKKbeta Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 ug/50 ul PEG20,000, Substrate: Rb CTF, 1.5 ug/50 ul, 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 ul reaction, increasing amounts of IKKbeta and 1.5 uM substrate peptide were used per reaction well at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-IKKbeta kinase fusion protien is 87 kDa (apparent molecular weight on SDS PAGE is 120 kDa). The purified kinase fusion protein was quality controlled for purity using SDS-PAGE Silver stain and Western blot [Fig.1]. IKKbeta kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure IKKbeta activity using HTScan™ IKKbeta Kinase Assay Kit #7549 [Fig.3].
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).
- Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.
- Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
- Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
- Brown, K. et al. (1995) Science 267, 1485-8.
- Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
- Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
- Chen, Z.J. et al. (1996) Cell 84, 853-62.
- Zandi, E. et al. (1997) Cell 91, 243-52.
- Karin, M. (1999) Oncogene 18, 6867-74.
- DiDonato, J.A. et al. (1997) Nature 388, 548-54.
- Mercurio, F. et al. (1997) Science 278, 860-6.
- Johnson, L.N. et al. (1996) Cell 85, 149-58.
- Delhase, M. et al. (1999) Science 284, 309-13.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 7549 HTScan® IKKβ Kinase Assay Kit
- 1146 IκB-α (Ser32) Biotinylated Peptide
- 9246 Phospho-IκBα (Ser32/36) (5A5) Mouse mAb
- 9802 Kinase Buffer (10X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
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.
