Our immobilized proteins (HPL-I, HUPA-I, HATIII-I, HTPA-I, SBTI-I, ELAS-I, ANHT-I) are manufactured using Bio-Rad brand Affi-Gel activated affinity chromatography media as the agarose matrix. These resins can be spun at 1000 g for 2-3 minutes. It is a soft gel and can be compressed (damaged) if centrifuged at higher speeds. These resins can be used repeatedly. After use, wash extensively with the original storage buffer including 0.02% azide to prevent bacterial growth and store at 4 C.
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.