Abstract
This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a microsprayer. We find a reproducible increase, rather than the expected decrease in GFP production as monitored by fluorescence, which may suggest heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios. The results have a bearing on the design and interpretation of validation experiments in time-resolved cryo-EM based on microfluidic chips.