Product Pathways - HTScan Kinase Assay Kits
HTScan® Ret Kinase Assay Kit #7773
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 |
| Gastrin Precursor (Tyr87) Biotinylated Peptide # 1310 | 1.25 milliliters |
| Ret Kinase # 7772 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human Ret kinase. It includes active Ret 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: 2,853Daltons. GST-Ret Kinase: 84 kDa.
Peptide Core Sequence
EAY*GW
Kinase Assay - Radiometric
Figure 1. Ret 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: PolyEY, 2 µg/50 µl, recombinant Ret: 200 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Ret kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by Ret kinase. In a 50 µl reaction, increasing amounts of Ret 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 Ret kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by Ret kinase. In a 50 µl reaction, 50 ng Ret, 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 Ret kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by Ret kinase. In a 50 µl reaction, 50 ng of Ret 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 Ret kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Ret substrate peptide (#1310) by Ret kinase. In a 50 µl reaction, 50 ng Ret 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 Ret (His658-Asp1110) (GenBank Accession No. NM_000323) 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 Mouse 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 Ret kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Ret 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 Ret activity using the Ret substrate peptide provided in this kit. Ret sensitivity to the inhibitor staurosporine was measured using the Ret substrate peptide provided in this kit [Fig.5].
Background
The Ret proto-oncogene (c-Ret) is a receptor tyrosine kinase that functions as a multicompetent receptor complex in conjunction with other membrane-bound ligand-binding GDNF family receptors (1). Ligands that bind the Ret receptor include the glial cell line-derived neurotropic factor (GDNF) and its congeners neurturin, persephin and artemin (2-4). Alterations in the corresponding Ret gene are associated with diseases including papillary thyroid carcinoma, multiple endocrine neoplasia (type 2A and 2B), familial medullary thyroid carcinoma and a congenital developmental disorder known as Hirschsprung’s disease (1,3). The Tyr905 residue located in the Ret kinase domain plays a crucial role in Ret catalytic and biological activity. Substitution of Phe for Tyr905 dramatically inhibits Ret autophosphorylation activity (5).
- Airaksinen, M.S. et al. (1999) Mol. Cell. Neurosci. 13, 313-325.
- Takahashi, M. et al. (1989) Oncogene 4, 805-806.
- Manie, S. et al. (2001) Trends Genet. 17, 580-589.
- Tallini, G. and Asa, S. (2001) Adv. Anat. Pathol. 8, 345-354.
- Iwashita, T. et al. (1999) Oncogene 18, 3919-3922.
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
- 7772 Ret Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
- 7450 Tyrosine Kinase Substrate Screening Kit
- 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.
