3 - Screening of Protein Crystallization Conditions on a...

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Unformatted text preview: Screening of Protein Crystallization Conditions on a Microfluidic Chip Using Nanoliter-Size Droplets Bo Zheng, L. Spencer Roach, and Rustem F. Ismagilov* Department of Chemistry, The Uni V ersity of Chicago, 5735 South Ellis A V enue, Chicago, Illinois 60637 Received July 9, 2003; E-mail: [email protected] This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization trial. Crystallization trials were set up inside 7.5-nL aqueous droplets. These droplets, each containing solutions of protein, precipitants, and additives in variable ratios, were formed in the flow of immiscible fluids inside microfluidic channels. 1,2 We have used the system to set up hundreds of trials at a rate of several trials per second under computer control. The goal of this Communication is to quantify this approach and validate it by crystallizing correct polymorphs of several common water- soluble proteins. New methods of protein crystallization are becoming especially important because of the success of genome sequencing projects. Crystallization is a bottleneck in determining tertiary protein structures from sequence data. 3 Protein crystallization occurs in the labile region of the crystallization phase diagram, a narrow region where nucleation but not precipitation can occur. 4 The phase diagram is multidimensional and complex, and, despite progress in theory, 5 concentrations of the protein and the reagents (precipi- tants, buffers, and additives) that place the solution into the labile region are usually determined by screening. Minimal volumes of the protein solution should be used during screening, because many proteins are only available in very small quantities. 6 Manual screening by mixing stock solutions in many ratios is time-consuming and requires at least 100 nL of the protein solution per trial. To overcome these limitations, robotic systems have been developed that can perform automated mixing of stock solutions, and which can set up crystallization trials with volumes from 1 μ L down to 100 nL, 7 consuming as little as 10 nL of individual solutions. 8 These robotic systems are expensive and have not yet seen wide adoption in individual laboratories. Microfluidic systems are useful for experiments that require minimal use of reagents. 9 Microfluidic platforms, therefore, are an attractive choice for macromolecular crystallization, 6 as was clearly demonstrated by Hansen et al. 10 These authors have crystallized proteins on a microfluidic device by free interface diffusion, a method that was previously possible only in microgravity. 10 Only ∼ 10 nL of the protein solution was used for each of 144 trials, which were conducted inside microfabricated chambers controlled by pressure-operated valves....
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This note was uploaded on 07/07/2010 for the course CHBE 471 taught by Professor Kraft during the Spring '08 term at University of Illinois at Urbana–Champaign.

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3 - Screening of Protein Crystallization Conditions on a...

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