Product Pathways - HTScan Kinase Assay Kits
HTScan® HER4/ErbB4 Kinase Assay Kit #7725
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-Tyrosine Mouse mAb (P-Tyr-100) # 9411 | 30 microliters |
| HTScan® Tyrosine Kinase Buffer (4X) # 9805 | 15 milliliters |
| DTT (1000x, 1.25 M) | 80 microliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| HER4/ErbB4 Kinase # 7724 | 5 micrograms |
| PYK2 (Tyr402) Biotinylated Peptide # 1315 | 1.25 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human HER4/ErbB4 kinase. It includes active HER4/ErbB4 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: 2,166 Daltons. GST-HER4/ErbB4 Kinase: 102 kDa.
Peptide Core Sequence
DIY*AE
Kinase Assay - Radiometric
Figure 1. HER4/ErbB4 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: PolyAEKY, 5 µg/50 µl, recombinant HER4/ErbB4: 50 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of HER4/ErbB4 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by HER4/ErbB4 kinase. In a 50 µl reaction, increasing amounts of HER4/ErbB4 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 HER4/ErbB4 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of HER4/ErbB4 substrate peptide (#1315) by HER4/ErbB4 kinase. In a 50 µl reaction, 50 ng HER4/ErbB4, 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 HER4/ErbB4 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by HER4/ErbB4 kinase. In a 50 µl reaction, 50 ng of HER4/ErbB4 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 HER4/ErbB4 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of HER4/ErbB4 substrate peptide (#1315) by HER4/ErbB4 kinase. In a 50 µl reaction, 50 ng HER4/ErbB4 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 a fragment of human HER4/ErbB4 (Arg676-Val1308) 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 Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified HER4/ErbB4 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the HER4/ErbB4 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 HER4/ErbB4 activity using the HER4/ErbB4 substrate peptide provided in this kit. HER4/ErbB4 sensitivity to the inhibitor staurosporine was measured using the HER4/ErbB4 substrate peptide provided in this kit [Fig.5].
Background
The HER/ErbB receptor tyrosine kinase family has been implicated in normal development, cardiac function and cancer (1,2). HER4/ErbB4, like other family members, has four ectodomains, a single transmembrane domain and a cytoplasmic tail containing the active tyrosine kinase domain (3). By binding to neuregulins and/or EGF family ligands, ErbB4 forms either a homodimer or heterodimer with other ErbB family members, which results in receptor activation and signaling (3). ErbB4 is ubiquitously expressed with the highest expression occurring in brain and heart. The expression of ErbB4 in breast cancer, pediatric brain cancer and other types of carcinomas has been reported suggesting that ErbB4 expression is involved in both normal tissue development and carcinogenesis (3).
- Holbro, T. and Hynes, N.E. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 195-217.
- Roskoski Jr, R. (2004) Biochem. Biophys. Res. Commun. 319, 1-11.
- Carpenter, G. (2003) Exp. Cell Res. 284, 66-77.
Application References
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