期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 103, 期 51, 页码 19243-19248出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0607502103
关键词
droplets; plugs; protein structure; high-throughput; miniaturization
资金
- NCI NIH HHS [Y1-CO-1020] Funding Source: Medline
- NCRR NIH HHS [P41 RR007707, RR07707] Funding Source: Medline
- NIGMS NIH HHS [R01 GM075827-01, Y1-GM-1104, U54 GM074961, R01 GM075827] Funding Source: Medline
High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a hybrid droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approximate to 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximate to 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 mu l of protein solution, approximate to 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据