Product Pathways - HTScan Kinase Assay Kits

HTScan® EGF Receptor Kinase Assay Kit #7909

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan^® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Kit Includes	Quantity
Phospho-Tyrosine Mouse mAb (P-Tyr-100) # 9411	30 microliters
HTScan^® Tyrosine Kinase Buffer (4X) # 9805	15 milliliters
DTT (1000x, 1.25M)	80 microliters
ATP (10 mM) # 9804	1 milliliters
PTP1B (Tyr66) Biotinylated Peptide # 1325	1.25 milliliters
EGF Receptor Kinase	5 micrograms

Description

The kit provides a means of performing kinase activity assays with recombinant human EGFR kinase. It includes active EGFR kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.

Molecular Weights

Peptide substrate, Biotin-PTP1B (Tyr66): 2141 Daltons. GST-EGFR Kinase: 87 kDa.

Peptide Core Sequence

DNDY*INA

Kinase Assay - Radiometric

Figure 1. EGFR kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 100 µM ATP, 250 µM PTP1B (Tyr66) Biotinylated Peptide (#1325) and variable amount of Recombinant EGFR. Reaction mixture incubated at room temperature for 10 minutes.

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of EGFR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1325) by EGFR kinase. In a 50 µl reaction, increasing amounts of EGFR and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Kinase Assay - DELFIA

Figure 5. Staurosporine inhibition of EGFR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of EGFR substrate peptide (#1366) by EGFR kinase. In a 50 µl reaction, 50 ng EGFR, 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.)

Kinase Assay - DELFIA

Figure 4. Peptide concentration dependence of EGFR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1325) by EGFR kinase. In a 50 µl reaction, 50 ng of EGFR and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Kinase Assay - DELFIA

Figure 2. Time course of EGFR kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of EGFR substrate peptide (#1325) by EGFR kinase. In a 50 µl reaction, 50 ng EGFR and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

Source / Purification

The GST-kinase fusion protein was produced using a baculovirus expression system with a construct expressing human EGFR (His672-Ala1210) (GenBank Accession No. NM_005228) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Quality Control

The substrate peptide was selected by using our Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.Purified EGFR kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. EGFR kinase activity was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify EGFR activity using the EGFR substrate peptide provided in this kit. EGFR sensitivity to the inhibitor staurosporine was measured using the EGFR substrate peptide provided in this kit [Fig.5].

Background

The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling 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 c-Cbl, an adaptor protein that leads 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 residues (Tyr1148 and Tyr1173) provides 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; mutations to either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

7450 Tyrosine Kinase Substrate Screening Kit
9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
1325 PTP1B (Tyr66) Biotinylated Peptide
9953 Staurosporine

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.