Fig. 3.

Impurities promote aggregation of lnC₂AB in solution. (A) Gel filtration profile on a Superdex 75 16/60 column of an lnC₂AB sample that was purified by our usual procedure, including the benzonase treatment, but without ion exchange chromatography step. (Inset) Peaks 1 and 2, as well as a control sample of fully purified lnC₂AB (pure), were analyzed by SDS/PAGE and Coomassie blue staining. (B) Analysis of lnC₂AB aggregation using a turbidity assay. The turbidity as monitored from the absorption at 400 nm was measured as a function of protein concentration for the same control sample of purified lnC₂AB (black circles), and for the samples from peaks 1 (blue squares) and 2 (red triangles) of the gel filtration experiment shown in A. The experiments were performed in 25 mM Tris (pH 7.4) containing 125 mM NaCl and 1 mM Ca²⁺. (C) UV spectra of samples corresponding to peaks 1 and 2 in the gel filtration chromatogram shown in A.