Product Pathways - HTScan Kinase Assay Kits
HTScan® IRAK4 Kinase Assay Kit #7552
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-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody # 3141 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide # 1344 | 1.25 milliliters |
| IRAK4 Kinase # 7551 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human IRAK4 kinase. It includes active IRAK4 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-peptide: 1,958 Daltons. GST-IRAK4 Kinase domain: 70 kDa
Peptide Core Sequence
YKT*LR
Kinase Assay - Radiometric
Figure 1. IRAK4 kinase activity was measured in a radioisotopic filter binding 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: Histone H2B, 5 µg/50 µl, Recombinant IRAK4: 20 ng/50 µl.
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 µl reaction, increasing amounts of IRAK4 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 IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of IRAK4 substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, 50 ng IRAK4, 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 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 µl reaction, 50 ng of IRAK4 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 IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of IRAK4 substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, 50 ng IRAK4 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 IRAK4 (Ala104-Ser460) (GenBank Accession No. AF445802.1) 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-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified IRAK4 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. IRAK4 kinase Vmax and Km values were 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 IRAK4 activity using the IRAK4 substrate peptide provided in this kit. IRAK4 sensitivity to the inhibitor staurosporine was measured using the IRAK4 substrate peptide provided in this kit [Fig.5].
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
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
- 3141 Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody
- 1344 Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide
- 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.
