EGFR Protein, Human (Active, sf9, His-GST)

EGFR is a transmembrane glycoprotein. EGFR binds to ligands, triggering receptor dimerization, inducing intracellular TK domain autophosphorylation, activating downstream Ras/MAPK, PI3K/Akt and other signaling pathways, and regulating cell proliferation, survival, migration and angiogenesis. EGFR Protein, Human (sf9, His-GST) is a recombinant EGFR protein expressed by Sf9 insect cells with N-His, N-GST tags.

EGFR belongs to the epidermal growth factor receptor family^{[1][2][3][4][5]}. EGFR is a transmembrane glycoprotein. EGFR has an extracellular domain of 621 amino acids, a single transmembrane domain of 23 amino acids, and an enzymatically active intracellular domain of 542 amino acids^[6]. EGFR binds to its ligand, triggers receptor dimerization, induces autophosphorylation of the intracellular TK domain, activates downstream Ras/MAPK, PI3K/Akt and other signaling pathways, and regulates cell proliferation, survival, migration and angiogenesis^[1][3][4][5]. EGFR is expressed in a variety of normal epithelial tissues and tumor tissues, such as breast, lung, stomach, colon, head and neck, and its abnormal expression is associated with the malignant progression of tumors^{[1][2][3][4][5]}.

The specific activity was determined to be ≥4 nmol/min/mg using Poly(Glu:Tyr) 4:1 as substrate.

Materials
Assay buffer A(5X): 200 mM Tris-HCl, pH 7.5, 100 mM MgCl2 and 0.5 mg/mL BSA
Buffer 1(2X): 1 mL assay buffer A, 2.5 μL DTT (0.1 M), 10 μL MnCl₂ (1 M) and 1.5 mL of dH₂0
Buffer 2 (1X): 1 mL assay buffer 1, and 1 mL dH₂O
Test protein: EGFR Protein, Human (Active, sf9, His-GST) (HY-P72987)
Substrate: Poly (Glu : Tyr) 4:1
ADP-Glo kit

Procedure
1. Thaw the ADP-Glo^TM Reagents at ambient temperature. Then prepare Kinase Detection Reagent by mixing Kinase assay buffer with the Lyophilized Kinase Detection Substrate.
2. Thaw the components of enzyme system, ADP and ATP on ice.
3. Prepare 1 mL of 250 μM ATP Sssay Solution by adding 25 μL ATP solution (10 mM) to 500 μL of 2X buffer and 475 μL of dH₂O.
4. In a white 96 well plate, add the following reaction components bringing the initial reaction volume up to 20 μL:
a. 10 μL of diluted active enzyme.
b. 5 μL of 1mg/mL stock solution of substrate (final Conc.: 0.2 mg/mL).
5. Set up the blank control as outlined in step (4), excluding the addition of the substrate. Replace the substrate with an equal volume of dH₂O.
6. Initiate the reactions by the addition of 5 μL of 250 μM ATP assay solution thereby bringing the final volume up to 25 μL. Shake the plate and incubate the reaction mixture at R.T. for 60 minutes.

Human

Sf9 insect cells

N-GST;N-10*His

P00533-1 (M668-A1210)

1956  [NCBI]

Cytoplasmic Domain

MRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

Approximately 89.1 kDa, based on SDS-PAGE under reducing conditions.

≥ 75%, as determined by reducing SDS-PAGE.

Solution.

1.Supplied as a 0.22 μm filtered solution of 20 mM Tris, 500 mM NaCl, 10 % glycerol, pH 7.4.
2.Supplied as a 0.22 μm filtered solution of 20 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.5, 0.02% Triton X-100, 0.4 mM β-mercaptoethanol, 0.5 mM PMSF, 2 mM GSH.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.
Please refer to the lot-specific COA for specific buffer information.

<1 EU/μg, determined by LAL method.

Please use rapid thawing with running water to thaw the protein.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. Lemmon M A, et al. Cell signaling by receptor tyrosine kinases. Cell, 2010, 141(7): 1117-1134.

  [2]. Giaccone G. HER1/EGFR-targeted agents: predicting the future for patients with unpredictable outcomes to therapy. Ann Oncol. 2005 Apr;16(4):538-48.  [Content Brief]

  [3]. Yarden Y, et al. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001 Feb;2(2):127-37.  [Content Brief]

  [4]. Shepard HM, et al. Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family. J Clin Invest. 2008 Nov;118(11):3574-81.  [Content Brief]

  [5]. Okines A, et al. Targeting the human EGFR family in esophagogastric cancer. Nat Rev Clin Oncol. 2011 Apr 5;8(8):492-503.  [Content Brief]

  [6]. Ho ECH, et al. Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor'. Antib Ther. 2019 Oct;2(4):88-98.  [Content Brief]