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

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.

Components	Volume (μL)	Blank Control (μL)
Active enzyme	10	10
Substrate stock solution (1 mg/mL)	5	5 μL dH2O
2X buffer	5	5
ATP assay solution (250 μM)	5	5

7. As the kinase reaction, set up an ATP to ADP conversion curve at 50 μM ATP/ADP range as described in the ADP-Glo^TM Kinase Assay technical Manual.
8. Terminate the reaction and deplete the remaining ATP by adding 25 μL of ADP- GloTM Reagent. Shake the 96 well plate and then incubate the reaction mixture for another 40 minutes at R.T.
9. Add 50 μL of the Kinase Detection Reagent, shake the plate and then incubate the reaction mixture for another 30 minutes at R.T.
10. Read the 96-well reaction plate using Microplate Luminomet.
11. Calculate specific activity:

Specific Activity (nmol/min/μg) =	Phosphate released* (nmol)
	Reaction time (min） × amount of enzyme (μg)

*Derived from the phosphate standard curve and adjusted for Control.