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Encompassing Amino Acids: 669-1022
Application: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: EGFR (V948R) activity was measured by using Poly (4:1 Glu, Tyr) synthetic peptide substrate diluted in 25 mM Tris-HCl buffer (pH 7.5) to a final concentration of 1 mg/ml in the ADP Glo™ assay (Promega #V9101). Reaction was initiated by mixing increasing amounts of the EGFR (V948R) with 25 µM ATP in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml BSA prepared with 250 µM DTT and the 20 µg/ml substrate.After a 40-minute incubation at 37°C, the reaction was terminated by addition of the AMP-Glo™ Reagent followed by a subsequent 40-minute incubation at room temperature. Kinase Detection Reagent was then added and incubated for another 30 minutes. Detection of luminescence was measured using the Luminescence Module Protocol on GloMax®-Multi Microplate reader. The corrected activity (RLU) was calculated by removing the blank value for each sample divided by the (specific activity of ADP in RLU/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg). The blank was determined from a 'no kinase' sample by replacing the enzyme working solution with an equal volume of Kinase Dilution Buffer X (1X).
Description: Recombinant human EGFR (epidermal growth factor receptor), encompassing amino acids (699-1022) length. This construct contains an N-terminal His-tag and mutation of interest (V948R). The recombinant protein was affinity purified and is kinase active.
Format: Aqueous buffer solution
Formulation: 50 mM Sodium Phosphate, pH 7.0, 300 mM NaCl, 150 mM Imidazole, 0.25 mM DTT, 25% glycerol
Genbank: NM_005228
Host Cell Line: Sf9 cells
Purity: ≥90%
Tags: N-terminal His-tag
Uniprot: P00533
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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