Figure 4.

SDS-PAGE analysis of thrombin cleavage of FXIII activation peptide by thrombin. A) 1 µM FXIII V34X variant was incubated with 30 nM recombinant thrombin at 37°C. At defined time points, the aliquots were removed, quenched with PPACK and then reducing loading buffer, and finally quickly frozen at −80°C. Samples were later subjected to electrophoresis on 8% gels and stained for proteins using Coomassie blue. Intact FXIII appears at 83kDa whereas cleavage of the Activation Peptide (1–37) results in a protein band at 79kDa. B) Cleavage progress for FXIII L34 (red), V34 (yellow), F34 (purple), Y34 (green), and W34 (blue) was monitored by densitometric analysis using GelAnalyzer 2010 (www.gelanalyzer.com) for the time points 1, 2, 5, 10, 15, 20, 25, and 30 min