NDLI: Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.

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Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.

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Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Li, Dianfan Boland, Coilín Walsh, Kilian Caffrey, Martin
Description	Country affiliation: Ireland Author Affiliation: Li D ( Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College Dublin, Ireland.)
Abstract	Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group(1-3). JoVE video articles describing the method are available(1,4). The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group(5,6). The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/4000
Journal	Journal of Visualized Experiments
Issue Number	67
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-09-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Crystallization Instrumentation Lipids Chemistry Membrane Proteins Robotics High-throughput Screening Assays Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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