Product Pathways - HTScan Kinase Assay Kits
HTScan® HER2/ErbB2 Kinase Assay Kit #7058
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 |
| FLT3 (Tyr589) Biotinylated Peptide # 1305 | 1.25 milliliters |
| HER2/ErbB2 Kinase # 7382 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human HER2/ErbB2 kinase. It includes active HER2/ErbB2 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-FLT3 (Tyr589) peptide: 1,945 Daltons. GST-HER2/ErbB2 Kinase: 116 kDa.
Peptide Core Sequence
NEY*FY*V
Kinase Assay - Radiometric
Figure 1. HER2/ErbB2 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 5 mM MnCl2, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Poly(Glu-Tyr), 400 ng/μL, and recombinant HER2/ErbB2: variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of HER2/ErbB2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by HER2/ErbB2 kinase. In a 50 µl reaction, increasing amounts of HER2/ErbB2 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 HER2/ErbB2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of HER2/ErbB2 substrate peptide (#1305) by HER2/ErbB2 kinase. In a 50 µl reaction, 100 ng HER2/ErbB2, 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 HER2/ErbB2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by HER2/ErbB2 kinase. In a 50 µl reaction, 50 ng of HER2/ErbB2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
DELFIA
Figure 2. Time course of HER2/ErbB2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of HER2/ErbB2 substrate peptide (#1305) by HER2/ErbB2 kinase. In a 50 µl reaction, 50 ng HER2/ErbB2 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 HER2/ErbB2 (Lys676-Val1255) (GenBank Accession No. NM_004448) 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 HER2/ErbB2 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. The specific activity of the HER2/ErbB2 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 HER2/ErbB2 activity using the HER2/ErbB2 substrate peptide provided in this kit. HER2/ErbB2 sensitivity to the inhibitor staurosporine was measured using the HER2/ErbB2 substrate peptide provided in this kit [Fig.5].
Background
The ErbB2 (HER2) proto-oncogene encodes a 185 kDa transmembrane, receptor-like glycoprotein with intrinsic tyrosine kinase activity (1). While ErbB2 lacks an identified ligand, ErbB2 kinase activity can be activated in the absence of a ligand when overexpressed and through heteromeric associations with other ErbB family members (2). Amplification of the ErbB2 gene and overexpression of its product are detected in almost 40% of human breast cancers (3). Binding of the c-Cbl ubiquitin ligase to ErbB2 at Tyr1112 leads to ErbB2 poly-ubiquitination and enhances degradation of this kinase (4). ErbB2 is a key therapeutic target in the treatment of breast cancer and other carcinomas with the regulation of ErbB2 degradation by the c-Cbl-regulated proteolytic pathway as one potential therapeutic strategy.Phosphorylation of the kinase domain residue Tyr877 of ErbB2 (homologous to Tyr416 of pp60c-Src) may be involved in regulating ErbB2 biological activity. The major autophosphorylation sites in ErbB2 areTyr1248 and Tyr1221/1222; phosphorylation of these sites couples ErbB2 to the Ras-Raf-MAP kinase signal transduction pathway (1,5).
- Muthuswamy, S.K. et al. (1999) Mol. Cell. Biol. 19, 6845-6857.
- Qian, X. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 1500-1504.
- Dittadi, R. and Gion, M. (2000) J. Natl. Cancer Inst. 92, 1443-1444.
- Klapper, L.N. et al. (2000) Cancer Res. 60, 3384-3388.
- Kwon, Y.K. et al. (1997) J. Neurosci. 17, 8293-8299.
Application References
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