Product Pathways - NF-kappaB Signaling
IRAK4 Kinase #7551
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 human IRAK4 kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human IRAK4 (Ala104-Ser460) (GenBank Accession No. AF445802.1) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography purification using glutathione-agarose.
Gel Staining
Figure 1. The purity of the IRAK4 fusion protein was analyzed using SDS/PAGE followed by anti-IRAK4 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. IRAK4 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: Histone H2B, 5 ug/50 ul, Recombinant IRAK4: 20 ng/50 ul.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of substrate peptide (#1344) by IRAK4 kinase. In a 50 ul reaction, increasing amounts of IRAK4 and 1.5 uM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-IRAK4 fusion protein is 70 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. IRAK4 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure IRAK4 activity using HTScan™ IRAK4 Kinase Assay Kit #7552 [Fig.3].
Background
Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is a serine/threonine-specific kinase that can be coprecipitated in an IL-1-inducible manner with the IL-1 receptor (1). The mammalian family of IRAK molecules contains four members (IRAK1, IRAK2, IRAK3/IRAK-M and IRAK4). The binding of IL-1 to IL-1 receptor type I (IL-1RI) initiates the formation of a complex that includes IL-1RI, AcP, MyD88 and IRAKs (2). IRAK undergoes autophosphorylation shortly after IL-1 stimulation. The subsequent events involve IRAK dissociation from the IL-1RI complex, its ubiquitination and its association with two membrane-bound proteins: TAB2 and TRAF6. The resulting IRAK-TRAF6-TAB2 complex is then released into the cytoplasm and activates protein kinase cascades, which include TAK1, IKKs and the stress-activated kinases (3).
- Dinarello, C.A. (1996) Blood 87, 2095-2147.
- Takaesu, G. et al. (2001) Mol. Cell. Biol. 21, 2475-2484.
- Janssens, S. and Beyaert, R. (2003) Mol. Cell 11, 293-302.
Application References
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Companion Products
- 1344 Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide
- 3141 Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody
- 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.
