Revision 1

#7773Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P07949

Entrez-Gene Id:

5979

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol. Store at –80°C.
Keep enzymes on ice during use. Avoid repeated freeze-thaw cycles.

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.

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

Background

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 1
#7773

HTScan® Ret Kinase Assay Kit

HTScan® Ret Kinase Assay Kit: Image 1 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用数量递增的 Ret 和 1.5 µM 底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Ret Kinase Assay Kit: Image 2 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng Ret、1.5 µM 底物肽、20 µM ATP 和数量递增的 staurosporine 反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Ret Kinase Assay Kit: Image 3 Expand Image
图 1. 使用以下反应条件在放射测定法中测量 Ret 激酶活性： 60 mM HEPES-NaOH、pH 7.5、3 mM MgCl2、3 mM CaCl2、1 mM EDTA、3 µM 原钒酸钠、1.2 mM DTT、ATP（变量）、2.5 µg/50 µl PEG20,000，底物：PolyEY、2 µg/50 µl、重组 Ret：200 ng/50 µl。
HTScan® Ret Kinase Assay Kit: Image 4 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng Ret 和浓度递增的底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® Ret Kinase Assay Kit: Image 5 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔用 50 ng Ret 和 1.5 µM 底物肽。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。