A high-throughput robotic system has been developed for crystallizing membrane proteins using lipidic mesophases. It incorporates commercially available components and is relatively inexpensive. The crystallization robot uses standard automated liquid-handlers and a specially built device for accurately and reproducibly delivering nanolitre volumes of highly viscous protein/lipid mesophases. Under standard conditions, the robot uses just 20 nl protein solution, 30 nl lipid and 1 microl precipitant solution. 96 wells can be set up using the robot in 13 min. Trials are performed in specially designed 96-well glass plates. The slim (<2 mm high) plates have exquisite optical properties and are well suited for the detection of microcrystals and for birefringence-free imaging between crossed polarizers. Quantitative evaluation of the crystallization progress is performed using an automated imaging system. The optics, in combination with the slim crystallization plates, enables in-focus imaging of the entire well volume in a single shot such that a 96-well plate can be imaged in just 4.5 min. The performance characteristics of the robotic system and the versatility of the crystallization robot in performing vapor-diffusion, microbatch and bicelle crystallizations of membrane and soluble proteins are described.
Abstract-A high-throughput robotic system has been developed to enable the automatic handling of nanoliter volumes of highly viscous biomaterials for crystallizing membrane proteins using lipidic mesophases. The in meso method introduced a few years ago has produced crystal structures of a number of important membrane proteins. The bottleneck to achieve high throughput automation of this, so-called in meso method, is the handling of nanoliter volumes of the highly viscous cubic phase as part of the crystallization process. The cubic phase sticks to everything that it has contact with and has a tendency to disable dispensing robotic tools. In this paper, we discuss the factors that influence the successful and automatic delivery of nanoliter volumes of the cubic phase. A creative cubic-phase-based coordinate measuring mechanism is presented for controlling the dispensing distance of the cubic phase which is critically important for the successful performance of the system. A mathematical model describing the cubic-phase delivery is proposed and verified. We also present the optimization of liquid handling parameters for the successful and automatic delivery of different precipitant solutions. The performance characteristics of the robotic system in terms of accuracy and reproducibility of delivering nano volumes of highly viscous biomaterials and micro volumes of different precipitant solutions are reported.Note to Practitioners-Automatic handling of nanoliter volumes of highly viscous biomaterials is a practically challenging yet important task in research-and-development activities of biology and drug industry, including protein crystallization. The latter, however, is a key step for determining the structure of the proteins. Only when the structure of the proteins is revealed, can one understand the function of the proteins and/or develop drugs to cure various kinds of diseases. This paper uses a practical project, crystallizing membrane proteins using lipidic mesophases, as an example to develop approaches for automatically handling highly viscous biomaterials. This paper presents the factors that influence the successful and automatic delivery of nanoliter volumes of viscous bio-
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.