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7430
HTScan® INS Receptor Kinase Assay Kit

HTScan® INS Receptor Kinase Assay Kit #7430

This product is discontinued

Kinase Assay - Radiometric Image 1

Figure 1. INSR 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: PolyAEKY, 10 µg/50 µl, Recombinant INSR: 25 ng/50 µl.

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Kinase Assay - DELFIA Image 2

Figure 3. Dose dependence curve of INSR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1325) by INSR kinase. In a 50 µl reaction, increasing amounts of INSR 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 INSR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of INSR substrate peptide (#1320) by INSR kinase. In a 50 µl reaction, 50 ng INSR, 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 INSR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1320) by INSR kinase. In a 50 µl reaction, 50 ng of INSR 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 INSR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of INSR substrate peptide (#1320) by INSR kinase. In a 50 µl reaction, 50 ng INSR 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 INSR kinase. It includes active INSR 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- IRS1 (Tyr891) peptide: 2,005 Daltons. GST-INSR Kinase: 70 kDa.

Peptide Core Sequence:

GEY*VN

Insulin receptor (INSR) is a membrane receptor tyrosine kinase. The receptor molecule consists of a disulfide linked heterodimer. The α subunit is a 135 kDa extracellular fragment, and the β subunit is a 95 kDa fragment containing an extracellular domain, a single transmembrane domain, and an intracellular tyrosine kinase domain (1). Insulin ligand binding to this receptor results in receptor autophosphorylation and tyrosine kinase activation. INSR catalyzes the tyrosine phosphorylation of molecules such as IRS, Gab1, Shc, and Cbl, which further activate the downstream MAPK, PI3K, and TC10 pathways. This eventually leads to increases in glucose uptake and metabolism as well as cell growth (2,3). INSR has peptide substrate specificity similar to other receptor tyrosine kinase members, preferring acidic residues at the -1 to -4 positions and large hydrophobic amino acids at positions +1 and +3 (4).

  1. Yip, C.C. and Ottensmeyer, P. (2003) J. Biol. Chem. 278, 27329-27332.
  2. Saltiel, A.R. and Pessin, J.E. (2002) Trends Cell Biol. 12, 65-71.
  3. Zick, Y. (2001) Trends Cell Biol. 11, 437-441.
  4. Songyang, Z. and Cantley, L.C. (1995) TIBS 20, 470-475.
Entrez-Gene Id
3643
Swiss-Prot Acc.
P06213
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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