Normalization scheme (Invariant Residue Normalization) applied to a Mg2+ titration of the P4-P6 subdomain of the T. thermophila group I intron. In this case four invariant residues were used to normalize the data (146 147 148 and 195) determined according to. A) (Top Pane) Raw peak areas as determined by SAFA for lanes corresponding to Mg2+ concentrations of 0.1, 0.3, 6, 11, and 100 mM (Green, Blue, Red, Cyan and Magenta, respectively). (Bottom Pane) Data normalized using invariant residue normalization procedure. A reference lane (in this case the Green data (0.1 mM Mg2+) is used and then a scaling factor is computed for all the other data such that the differences of the invariant peaks are minimized. B) Titration curves for Residue 182 and 213 based on the data in A showing the Mg2+ dependence of these two protections. A Hill model is fit to the data with a cooperativity value (n=3) to obtain a midpoint of 1.7 and 1.9 mM Mg2+ for residues 182 and 213, respectively.