The tight control of nucleation in counterdiffusive flow regimes by hyperfluorinated oils

There is mounting evidence of the advantages of using oils to obtain protein crystals of good size and quality suitable for high-resolution X-ray crystallography. Most of the studies reported to date concern the use of oils in vapor diffusion and microbatch methods. In contrast, for crystallization methods that are based on counterdiffusion, very little is known about the effect of high-density oils on crystal growth. The present work is devoted to the systematic analysis of several fluorinated oils of high density on the nucleation and growth rate of protein crystals. The experimental setup includes a multi-cell crystallization reactor that combines the advantages of crystal growth inside capillary tubes with the incorporation of gels to ensure a counterdiffusive flow regime. The results show that fluorinated oils affect nucleation and crystal growth rate in a different degree and that the decrease of crystal growth rate is proportional to the extent of fluorination and oil density. This work provides a rational framework on the use of oils in protein crystallization when counterdiffusion is the method of choice. Moreover, the advantageous use of fluorinated oils can also be extended to other crystallization methods more widely used, including those based on vapor-diffusion equilibrium.