Recombinant Human EGFR Kinase Domain His-tag Protein, CF Summary
Product Specifications
Gly696-Gly1022, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
11142-ER
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Tris, 20 mM MgCl2, 5 mM MnCl2, 0.1 mg/mL BSA, pH 7.5
- Recombinant Human EGFR (rhEGFR) (Catalog # 11142-ER)
- Poly (4:1 Gly, Tyr), 1 mg/mL stock in 25 mM Tris, pH 7.5
- Adenosine triphosphate (ATP), 10 mM stock in deionized water
- ADP-GloTM Kinase Assay (Promega)
- White 96-well Plate
- Luminescent Plate Reader
- Dilute rhEGFR to 10 µg/mL in Assay Buffer.
- Prepare Substrate Mixture containing 65 µM ATP and 0.4 mg/mL Poly (4:1 Gly, Tyr) in Assay Buffer.
- Combine equal volumes of 10 µg/mL rhEGFR and Substrate Mixture. Replace enzyme with Assay Buffer for Substrate Control.
- Incubate at room temperature for 40 minutes.
- After incubation, transfer 10 µL of each reaction to a plate.
- Terminate the reaction and deplete the remaining ATP by adding 10 µL of ADP-GloTM Reagent to all wells.
- Incubate for 40 minutes at room temperature.
- Add 20 µL of Kinase Detection Reagent to all wells.
- Incubate at room temperature for 30 minutes.
- Read plate in Luminescence endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Luminescence* (RLU) x Conversion Factor** (pmol/RLU) |
| Incubation time (min) x amount of enzyme (µg) |
- rhEGFR: 0.05 µg
- ATP: 8.125 µM
- Poly (4:1 Gly, Tyr): 2 µg
Scientific Data
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2 μg/lane of Recombinant Human EGFR Kinase Domain His-tag Protein (Catalog # 11142-ER) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 39 kDa.
Reconstitution Calculator
Background: EGFR
The EGFR subfamily of receptor tyrosine kinases comprises four members: EGFR (also known as HER-1, ErbB1, or ErbB), ErbB2 (Neu, HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). All family members are type I transmembrane glycoproteins with an extracellular ligand binding domain containing two cysteine-rich domains separated by a spacer region and a cytoplasmic domain containing a membrane-proximal tyrosine kinase domain followed by multiple tyrosine autophosphorylation sites (1,2). The human EGFR cDNA encodes a 1210 amino acid (aa) precursor with a 24 aa signal peptide, a 621 aa extracellular domain (ECD), a 23 aa transmembrane segment, and a 542 aa cytoplasmic domain (3,4). The kinase domain contains the conserved catalytic domain with an ATP binding site in the cleft of two lobes at the N-terminal and C-terminal regions and an activation loop with a DFG motif that changes conformation to regulate activity (5-7). EGFR binds a subset of the EGF family ligands (1,2) that induce EGFR homodimerization as well as heterodimerization with other family members resulting in kinase activation and cell signaling (8-10). EGFR signaling regulates multiple biological functions including cell proliferation, differentiation, motility, and apoptosis (11,12). EGFR has been reported as overexpressed and/or mutated in tumors with high levels of activation of EGFR related to poor prognosis and tumor regression rates (13,14). EGFR kinase inhibition is a promising target for cancer therapeutics and includes development of drugs to address EGFR-mutation conferred resistance (7,15).
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