Cleaning protocols for crystallization robots: preventing protease contamination

Abstract

The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity.

The N14 anti-afamin antibody Fab: a rare VL1 CDR glycosylation, crystallographic re-sequencing, molecular plasticity and conservativeversusenthusiastic modelling

“…Crystals were grown at 291 K by sitting-drop vapourdiffusion in 96-well plates (Swissci 30926) using 200 nl droplets of antibody-fragment stock solution (10 mg ml À1 SEC-purified Fab fragment in 20 mM HEPES pH 7.5, 150 mM NaCl) mixed with 200 nl crystallization cocktail in a robotic setup using a Phoenix robot (Art Robbins Instruments, Sunnyvale, California, USA) equipped with a single nanoneedle protein dispenser (Krupka et al, 2002;Naschberger et al, 2015).…”