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7746
HTScan® IGF-1 Receptor Kinase Assay Kit

HTScan® IGF-1 Receptor Kinase Assay Kit #7746

This product is discontinued

Kinase Assay - Radiometric Image 1

Figure 1. IGF-IR 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 IGF-IR: 20 ng/50 µl.

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Kinase Assay - DELFIA Image 2

Figure 3. Dose dependence curve of IGF-IR kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1320) by IGF-IR kinase. In a 50 µl reaction, increasing amounts of IGF-IR 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 IGF-IR kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of IGF-IR substrate peptide (#1320) by IGF-IR kinase. In a 50 µl reaction, 50 ng IGF-IR, 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 IGF-IR kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1320) by IGF-IR kinase. In a 50 µl reaction, 50 ng of IGF-IR 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 IGF-IR kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of IGF-IR substrate peptide (#1320) by IGF-IR kinase. In a 50 µl reaction, 50 ng IGF-IR 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 IGF-IR kinase. It includes active IGF-IR 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: 2,005 Daltons. GST-IGF-IR Kinase: 77 kDa.

Peptide Core Sequence:

GEY*VN

Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Receptor autophosphorylation follows binding of the IGF-I and IGF-II ligands. Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).

  1. Adams, T.E. et al. (2000) Cell Mol Life Sci 57, 1050-93.
  2. Baserga, R. (2000) Oncogene 19, 5574-81.
  3. Scheidegger, K.J. et al. (2000) J Biol Chem 275, 38921-8.
  4. Hernández-Sánchez, C. et al. (1995) J Biol Chem 270, 29176-81.
  5. Lopaczynski, W. et al. (2000) Biochem Biophys Res Commun 279, 955-60.
  6. Baserga, R. (1999) Exp Cell Res 253, 1-6.
  7. White, M.F. et al. (1985) J Biol Chem 260, 9470-8.
  8. White, M.F. et al. (1988) J Biol Chem 263, 2969-80.
Entrez-Gene Id
3480
Swiss-Prot Acc.
P08069
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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