Mass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration-depleted solution surrounding the crystal. Finally, the counter-diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments.

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We report an efficient screening methodology based on the capillary counter-diffusion technique (CCD), which was evaluated using two different practical approaches. The first consisted of kits prepared with the most successful crystallizing agents (PEG and ammonium sulfate) buffered at different pHs ranging from 4 to 9 and tested on 14 samples, including commercial and research target proteins. The second approach was based on the previously identified and highly effective 24 crystallization cocktails adapted to the counterdiffusion setup. This screening was tested with two target proteins, HbII and HbII-III from the clam Lucina pectinate, and the results compared with those obtained with the vapor-diffusion experiment. The success rate was higher than 60% in both approaches. These results experimentally confirm the usefulness of the CCD technique for the screening of crystallization conditions of biomacromolecules beyond its well-known value for the growth of large and high-quality crystals. We describe a detailed protocol for the laboratory implementation of the capillary counter-diffusion technique.

In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.