Silverquest silver stain kit

The sample was trapped and cross-linked using the GraFix protocol^{23 (link)}. Equimolar quantities of SidJ E565A/CaM and SdeA were incubated on ice for 30 min in the presence of 150 mM NaCl, 50 mM HEPES, 10 mM MgCl2, and 10 mM ATP and loaded onto 5–20% glycerol gradients using 0–0.1% glutaraldehyde and 100 mM NaCl, 10 mM HEPES pH 7.5, 10 mM MgCl2, and the tubes were centrifuged at 164,000xg for 18 h at 4 °C. The gradients were subsequently fractionated and quenched through the addition of 10 mM Tris (final concentration). The fractions were evaluated by SDS-PAGE and silver staining using a SilverQuest Silver Stain Kit (Invitrogen). The fractions containing a band of the desired weight were pooled, concentrated, and loaded onto a pre-equilibrated (150 mM NaCl, 10 mM HEPES pH 7.5, 10 mM MgCl2, 0.5 mM TCEP) Superdex S200 3.2/300 size-exclusion column (GE Life Sciences). The peak fractions were pooled and concentrated to 0.3–0.5 mg/mL and used for grid preparation.