Crosslinking gradient (GraFix)

The elution fractions of the freshly purified complexes were pooled and transferred to a crosslinking gradient, GraFix (Kastner et al., 2008), with the following buffer composition: 10% - 30% Glycerol, 150 mM KCl, 20 mM HEPES KOH pH 7.8; 0,05% Glutaraldehyde and spun for 14 h at 160,000 g (SW60Ti). The gradient was aliquoted in 150 μl fractions, quenched with 20 mM Tris-HCl pH 7.8 (final concentration) and the protein complex traced with a Dot-Blot against the SBP-tag. Fractions containing cross-linked complex were pooled and the glycerol was removed by multiple rounds of concentration using an 0.5 mL Amicon spin column (50 kDa cut-off).