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Microelectrophoresis platform for fast serial analysis of single cells.

ABSTRACT: A capillary-based microelectrophoresis platform for fast serial analysis of single cells is described. In this system, the capillary remains fixed and a two-channel flow system is used to rapidly switch the buffer surrounding the capillary inlet from a physiological buffer to an electrophoretic buffer. Single cells are retained in the physiologic buffer channel utilizing an array of cell microwells patterned into the channel floor. The defined addresses of the cells on the array enable the sequential delivery of individual cells to the inlet of the capillary, where a focused laser pulse lyses the cell. The cell chamber is moved along a preordained route so that the inlet of the capillary is located in the electrophoresis buffer for separation and the physiological buffer during cell sampling. The throughput of the current system is limited by peak overlap between successive samples. Key characterizations of this system including the fluid flow rates, the cell array dimensions, and laser energies were performed. To demonstrate this system, 28 cells loaded with Oregon green and fluorescein were serially analyzed in under 16 min, a rate of 1.8 cells/min.

SUBMITTER: Jiang D

PROVIDER: S-EPMC2993077 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Microelectrophoresis platform for fast serial analysis of single cells.

Jiang Dechen D Sims Christopher E CE Allbritton Nancy L NL

Electrophoresis 20100801 15

A capillary-based microelectrophoresis platform for fast serial analysis of single cells is described. In this system, the capillary remains fixed and a two-channel flow system is used to rapidly switch the buffer surrounding the capillary inlet from a physiological buffer to an electrophoretic buffer. Single cells are retained in the physiologic buffer channel utilizing an array of cell microwells patterned into the channel floor. The defined addresses of the cells on the array enable the seque ...[more]

PMID: 20603824

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Project description:Summary Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Graphical abstract Highlights • A platform for rapid in meso structures by serial crystallography (SSX)• Insights into ADIPOR2 receptor-ligand dynamic interactions• A web-based application for remote user-guided experimental design and execution• An automated SSX-based ligand discovery pipeline for membrane proteins is introduced Motivation Membrane proteins are key regulators of most physiological processes and represent attractive targets for drug discovery programs. One of the most successful methods to obtain high-resolution structures of membrane proteins relies on in meso crystallization in combination with serial synchrotron crystallography. However, this remains a difficult process, with challenges at every step, including complex manual sample recovery protocols leading to limited throughput and sample loss and the difficulty in carrying out high-throughput ligand screening experiments. We have developed a new approach enabling rapid, automated structural analysis of membrane proteins in meso based on the CrystalDirect technology that addresses these issues, enabling high-throughput drug discovery with membrane proteins. Membrane proteins control many biological processes and represent attractive targets for drug discovery, but are difficult to study structurally. Healey et al. present an automated approach, combining the CrystalDirect technology and serial crystallography, for rapid structural analysis of membrane proteins and opening new opportunities for high-throughput drug discovery.

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Microelectrophoresis platform for fast serial analysis of single cells.

Publications

Microelectrophoresis platform for fast serial analysis of single cells.

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