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7773
HTScan® Ret Kinase Assay Kit

HTScan® Ret Kinase Assay Kit #7773

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
DTT (1000X) 1 x 80 µl
ATP (10 mM) 9804 1 x 1 ml

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 Formula:

Peptide substrate, Biotin-peptide: 2,853Daltons. GST-Ret Kinase: 84 kDa.

Peptide Core Sequence:

EAY*GW

The Ret proto-oncogene (c-Ret) is a receptor tyrosine kinase that functions as a multicomponent 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 neurotrophic factor (GDNF) and its congeners neurturin, persephin, and artemin (2-4). Research studies have shown that 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 Tyr at position 905 dramatically inhibits Ret autophosphorylation activity (5).

  1. Airaksinen, M.S. et al. (1999) Mol Cell Neurosci 13, 313-25.
  2. Takahashi, M. et al. (1989) Oncogene 4, 805-6.
  3. Manié, S. et al. (2001) Trends Genet 17, 580-9.
  4. Tallini, G. and Asa, S.L. (2001) Adv Anat Pathol 8, 345-54.
  5. Iwashita, T. et al. (1999) Oncogene 18, 3919-22.
Entrez-Gene Id
5979
Swiss-Prot Acc.
P07949
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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