To determine complete activation of plasminogen to plasmin with uPA, follow this protocol:
1. Prepare a 10% Tris-Glycine SDS-PAGE protein gel, or similar gel capable of resolving 50-100 kDa.
2. Prepare three tubes with 10ul of TBS, 1ug of HGPG, and 0.1ug of uPA.
3. Create three time points by adding 10ul of reducing sample loading buffer to tubes at 1min, 10min, and 30min.
4. Prepare one tube with 10ul of TBS, 1ug of HGPG, and 10ul of reducing sample loading buffer to create a negative control.
5. Boil all tubes for 1min.
6. Run all samples on SDS-PAGE and Coomassie stain gel.
7. The negative control will appear as a single band at 92 kDa.
8. The time points will appear as multiple bands starting at 85 kDa.
9. Conversion to plasmin is indicated by disappearance of the band at 92 kDa.
Did You Find This Article Helpful?
No - 0 visitors found this post was not helpful