PAI-1 Inhibition Kinetic Assay with uPA

PAI-1 Inhibition of uPA
1. uPA Control Rate – Incubate 5 ug of human uPA in a total volume of 100 ul TBS for 1 minute. Add 900 ul of Glu-Gly-Arg pNA urokinase substrate at 200 uM in TBS, mix and take kinetic absorbance at 405 nm for 1 minute. Record linear rate.
2. PAI-1 Inhibition Rates – Incubate 5 ug of uPA with 1 ug of sample PAI-1 in a total volume of 100 ul TBS for 1 minute. Add 900 ul of Glu-Gly-Arg pNA urokinase substrate at 200 uM in TBS, mix and take kinetic absorbance at 405 nm for 1 minute. Record linear rate. Repeat with 2 and 3 ug PAI-1.
3. Calculation of PAI-1 Activity – Plot Control and Inhibition rates with PAI-1 on X-axis and rate on Y-axis. Generate linear curve then solve linear equation for X by setting Y=0. Use the following equation to determine PAI-1 activity:
X / 5 * 54 / 43 * 100 = % Active PAI-1

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