Figure 3.

Fluid phase analysis of inactive (S525A) FI. (A) SDS-PAGE demonstrates a lack of proteolytic activity of IRES S525A FI across a range of timepoints (5 – 60 minutes). Activity was assessed by the ability of factor I in combination with its cofactor, FH, to cleave C3b to its inactive form, iC3b. C3b cleavage was indicated by the generation of the α1 (68kDa) and the two α2 (46kDa, 43kDa) bands as demonstrated by the WT FI (positive) control. (B) Lack of activity of S525A FI – densitometry analysis. Licor ImageStudio Lite with median background subtraction was utilised to perform densitometry analysis on the SDS-PAGE gels revealing no activity of the S525A FI. The density of the α’-chain was given as a ratio of the β-chain density before being normalised to a negative control (with no FI) to give a relative density of the α’-chain remaining for each reaction.