Recombinant Human EGFR Kinase Domain His-tag Protein, CF

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.

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human EGFR Kinase Domain His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer phosphate from adenosine triphosphate (ATP) to a synthetic peptide substrate.
  The specific activity is >35 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human EGFR protein
  Gly696-Gly1022, with an N-terminal Met and 6-His tag
• Accession #: P00533.2
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 38 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 39 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT.
• 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 Glu, 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
  1. Dilute rhEGFR to 10 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 65 µM ATP and 0.4 mg/mL Poly (4:1 Glu, Tyr) in Assay Buffer.
  3. Combine equal volumes of 10 µg/mL rhEGFR and Substrate Mixture. Replace enzyme with Assay Buffer for Substrate Control.
  4. Incubate at room temperature for 40 minutes.
  5. After incubation, transfer 10 µL of each reaction to a plate.
  6. Terminate the reaction and deplete the remaining ATP by adding 10 µL of ADP-GloTM Reagent to all wells.
  7. Incubate for 40 minutes at room temperature.
  8. Add 20 µL of Kinase Detection Reagent to all wells.
  9. Incubate at room temperature for 30 minutes.
  10. Read plate in Luminescence endpoint mode.
  11. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Luminescence* (RLU) x Conversion Factor** (pmol/RLU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Control
  **Derived using ADP-GloTM Kinase Assay Kit protocol (Promega)
  Per Well:
  • rhEGFR: 0.05 µg
  • ATP: 8.125 µM
  • Poly (4:1 Glu, Tyr): 2 µg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human EGFR Kinase Domain His-tag Protein, CF

• avian erythroblastic leukemia viral (v-erb-b) oncogene homolog
• cell growth inhibiting protein 40
• cell proliferation-inducing protein 61
• EC 2.7.10
• EC 2.7.10.1
• EGF R
• EGFR
• epidermal growth factor receptor (avian erythroblastic leukemia viral (v-erb-b)oncogene homolog)
• epidermal growth factor receptor
• ErbB
• ErbB1
• ERBB1PIG61
• HER1
• HER-1
• mENA
• Proto-oncogene c-ErbB-1
• Receptor tyrosine-protein kinase erbB-1

Background

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).

1. Singh, A.B. and R.C. Harris (2005) Cell. Signal. 17:1183.
2. Shilo, B.Z. (2005) Development 132:4017.
3. Lin, C. et al. (1984) Science 224:843.
4. Ullrich, A. et al. (1984) Nature 309:418.
5. Stamos, J. et. al. (2002) J. Biol. Chem. 277:46265.
6. Liu, Q. et al. (2013) Chem. Biol. 20:146.
7. Amelia, T. et al. (2022) Molecules 27:819.
8. Graus-Porta, D. et al. (1997) EMBO J. 16:1647.
9. Yarden, Y. et al. (2001) Nat. Rev. Mol. Cell Biol. 2:127. 
10. Lemmon, M.A. (2009) Exp. Cell Res. 315:638.
11. Sibilia, M. and E.F. Wagner (1995) Science 269:234.
12. Miettinen, P.J. et al. (1995) Nature 376:337.
13. Janne, P.A. et al. (2005) J. Clin. Oncol. 23:3227.
14. Yun, C.-H. et al. (2008) Proc. Nat. Acad. Sci. USA 105:2070.
15. Yu, J. et al. (2022) Front. Mol. Biosci. 9:847825.

Bioinformatics

• Uniprot:
  • P00533.2()