TIMP-2 Protein, Mouse (HEK293, His)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl?, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Test protein: TIMP-2 Protein, Mouse (HEK293, His) (HY-P71137)
Tool protein: MMP-2 Protein, Human (HEK293, His) (HY-P70268)
Activator for Human MMP-2: p-Aminophenylmercuric acetate (HY-148905, APAM), dissolved in 40 mM DMSO
Substrate: MOCAc-PLGL(Dpa)AR (HY-131498), dissolved in 2 mM DMSO

Procedure
1. Dilute Human MMP-2 to 100 μg/mL in assay buffer.
2. Add APMA to Human MMP-2 to a final concentration of 1 mM.
3. Incubate 100 μg/mL Human MMP-2 at 37°C for 1 hour to activate.
4. Dilute Mouse TIMP-2 protein with assay buffer to concentrations of 400 nmol/L, 200 nmol/L, 100 nmol/L, 50 nmol/L, 25 nmol/L, 12.5 nmol/L, 6.25 nmol/L, 3.13 nmol/L, and 1.56 nmol/L.
5. Dilute the Human MMP-2 activated with assay buffer to 2 μg/mL.
6. Experimental group: 20 μL of Mouse TIMP-2 protein at varying concentrations + 20 μL of 2 μg/mL Human MMP-2 protein;
Control group: 20 μL assay buffer + 20 μL, 2 μg/mL Human MMP-2 protein.
7. Incubate reaction mixtures at 37°C for 2 hours.
8. Dilute the reaction by adding 160 μL assay buffer.
9. Dilute the substrate to 20 μM in assay buffer.
10. Dispense 50 μL of the diluted mixture into a black microplate and add 50 μL substrate to initiate the reaction.
11. Read for 5 minutes in dynamic mode at excitation wavelength 320 nm and emission wavelength 405 nm.
12. Determine the 50% inhibitory concentration (IC₅₀) for Mouse TIMP-2 by plotting the RFU/min versus concentration relationship using 4-PL fitting.