Figure 3. Dose dependence curve of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody PY100 #9411 to detect phosphorylation of peptide C03-1727 by GST-EGF-R kinase. In a 50 ul reaction, increasing amounts of GST-EGF-R and 5 uM substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 11152. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 6. Iressa and staurosporine inhibition of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of EGF-R substrate peptide (C03-1727) by GST-EGF-R kinase. In a 50 ul reaction, 10 Units GST-EGF-R , 1.5 uM substrate peptide, 20 uM ATP and increasing amount of inhibitor (iressa or staurosprine) were used per reaction well at 25ºC for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of peptide C03-1727 by GST-EGF-R kinase. In a 50 ul reaction, 10 Units of GST-EGF-R and increasing concentrations of substrate peptide were used per reaction well at 25ºC for 30 minutes. Background reading is 8985. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Time course of EGF-R kinase activity: DELFIA® data generated using Phospho-Tyrosine Monoclonal Antibody P-Tyr-100 #9411 to detect phosphorylation of EGF-R substrate peptide by GST-EGF-R kinase. In a 50 ul reaction, 10 Units GST-EGF-R and 5 uM substrate peptide were used per reaction well. Background reading is 5144. (DELFIA® is a registered trademark of PerkinElmer, Inc.).
Figure 2. Treatment of Tyrosine Kinase Substrate Screening Kit #7450 with EGF-R Kinase. Positive control is in yellow and negative control is in blue. Several positive hits for EGF-R were identified using this approach (in red). The star indicates the peptide chosen for this HTScan® EGF-R Kinase Assay Kit.
Figure 5. EGF-R kinase activity was measured in a radioisotopic filtration assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 ug / 50 ul PEG20.000, Substrate:PolyEY, 10 ug/50 ul, Recombinant EGF-R: 5 Units/50 ul.
Quality Control: PTP1B peptide was selected by using Tyrosine Kinase Substrates Screening Kit #7450 to screen for EGF-R kinase substrates. Phospho-Tyrosine Monoclonal Antibody #9411 was used for detection (fig.2). The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.
Purified EGF-R kinase was quality controlled for purity using silver stain SDS-PAGE and Western blot. EGF-R kinase Vmax and Km values were measured to determine specific enzymatic activity (fig.5).
Assay conditions (time course [fig.1], kinase dose-dependence [fig.3], substrate dose-dependence [fig.4] and inhibitor sensitivity [fig.6]) for EGFR kinase activity were verified using the EGF-R substrate peptide provided in this kit.
The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).
Description: The kit provides a means of performing enzymatic assays with active human EGF-R kinase. It includes active EGF-R kinase (supplied as a GST fusion protein), a biotinylated substrate peptide and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Unit Definition: 10 Units is defined as the amount of EGF-R kinase required to maximally phosphorylate 75 pmol of PTP1B (Tyr66) #C03-1727 biotinylated substrate peptide in 30 minutes at 25ºC in a total reaction volume of 50 µl quantified by DELFIA®.
Peptide Core Sequence: NDY*IN
Molecular Weights: Peptide Substrate, Biotin-PTP1B (Tyr66): 2141 Daltons, GST-EGF-R Kinase domain: 91,163 Daltons
Storage: Antibodies are supplied in 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 glutathion and 20% glycerol. Store at -80ºC.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.
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