To test the uPA activity of your sample, we recommend the following protocol for a 1.0 ml microcuvette:
1. Add 900 ul of uPA chromogenic substrate at 100 uM in TBS or PBS
2. Add 5 ul of uPA sample to cuvette and mix well
3. Run kinetic protocol at 405 nm for 60 seconds
To determine the percentage of your uPA that is still active, we recommend incubating 0.5 – 1.0 ug of uPA with a five fold molar excess of active PAI-1 in a neutral pH buffer, then running the sample on SDS-PAGE to check amount of uPA-PAI-1 complex that is formed. If the uPA was kept in the provided storage buffer (0.1 M Acetate, 0.1 M NaCl, 1 mM EDTA, pH 5.0) and frozen at -20 C or colder, there should be very little loss of activity.
For cleavage of proteins, we recommend adding a catalytic amount of uPA (usually a 1:100 molar ratio of uPA to target protein) and incubating in a neutral pH buffer. The reaction will proceed more slowly at room temperature than at 37 C. You should run pilot assays at a very small scale taking samples for SDS-PAGE at time points to determine the ideal time and temperature needed for complete cleavage without excessive non-specific degradation.
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