HPL-I Activation of tPA

1. Wash the HPL-I with 0.1 M Hepes 0.1 M NaCl pH 7.4.
2. Measure the tPA activity by chromogenic substrate before adding beads.
3. Add some washed beads to the tPA solution. I would recommend 50 ul of beads, but you can adjust it higher or lower.
4. Turn the reaction tube end-over-end to keep the beads resuspended in tPA solution and incubate at room temperature.
5. Every 10 minutes let the beads settle by gravity or centrifugation, then remove a small sample and assay it.
6. The tPA activity should increase ~ 4x when it becomes all 2 chain.
7. After a maximal activity is reached, remove the solution from the beads.
8. Perform SDS-PAGE under non-reducing and reducing conditions to observe the 2 chains.

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