Product Pathways - Tyrosine Kinase/ Adaptors
Ret Kinase #7772
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 Ret kinase (His658-Asp1110), 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 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.
Gel Staining
Figure 1. The purity of the GST-Ret fusion protein was analyzed using SDS/PAGE followed by anti-Ret Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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, 5 µg/50 µl and 200 ng/50 µl Recombinant Ret.
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.)
Quality Control
The theoretical molecular weight of the GST-Ret fusion protein is 84 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Ret kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Ret activity using HTScan™ Ret Kinase Assay Kit #7773 [Fig.3].
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
- 7773 HTScan® Ret Kinase Assay Kit
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
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.
