Product Pathways - Glucose Metabolism
IGF-1 Receptor Kinase #7745
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Description
Purified recombinant human IGF-IR kinase (Met954-Cys1367), 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 IGF-IR (Met954-Cys1367) (GenBank accession No. NM_000875) 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-IGF-IR fusion protein was analyzed using SDS/PAGE followed by anti-IGF-IR Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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 and 20 ng/50 µl Recombinant IGF-IR.
Kinase Assay - DELFIA
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.)
Quality Control
The theoretical molecular weight of the GST-IGF-1R fusion protein is 77 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. IGF-IR kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure IGF-IR activity using HTScan™ IGF-I Receptor Kinase Assay Kit #7746 [Fig.3].
Background
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 insulin receptor is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation of Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires the triple tyrosine phosphorylation (8).
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- Baserga, R. et al. (2000) Oncogene 19, 5574-5581.
- Scheidegger, K.J. et al. (2000) J. Biol. Chem. 275, 38921-38928.
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- Lopaczynski, W. et al. (2000) Biochem. Biophys. Res. Commun. 279, 955-960.
- Baserga, R. et al. (1999) Exp. Cell Res. 253, 1-6.
- White, M.F. et al. (1985) J. Biol. Chem. 260, 9470-9478.
- White, M.F. et al. (1988) J. Biol. Chem. 263, 2969-2980.
Application References
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