Sucrose density gradient ultracentrifugation

Sucrose density gradients 2–25% (w/v) in 50 mM HEPES‐KOH pH 8.2, 150 mM NaCl were prepared in 12 mL ultracentrifugation tubes (Beckman Coulter) using the GraFix protocol,18 with 0.15% (v/v) glutaraldehyde in the 25% (w/v) sucrose buffer as the cross‐linking agent. Prepared gradients were left standing at 4°C for at least 1 h prior to usage. A glutaraldehyde‐free cushion of 600 µL of the 2% (w/v) sucrose buffer was placed on top of the gradients. The sample (200 µL) was then layered on top of the cushion and ultracentrifuged (186,000g, 4°C, 18 h). The gradients were fractionated as 200‐µL aliquots collected from the top, and each of these mixed with 20 µL of 1 M Tris pH 8.0 to deactivate remaining glutaraldehyde.