We developed a microfluidic "hybrid" method that combines sparse-matrix and optimization-gradient screening into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption (PNAS 2006 103: 19243-19248). Many distinct reagents were sequentially introduced as "large" plugs (~ 140 nL) into a microfluidic device. Large plugs were combined with a protein sample and a diluting buffer so that multiple ~10 nL plugs of each reagent were formed over a range of concentrations. The concentrations of each reagent were well-controlled by a computer subroutine and were indexed with plug size. We validated the hybrid method by demonstrating its applicability to two challenging problems: i) crystallization of model membrane proteins and ii) handling of detergent solublized membrane protein and viscous precipitants. We overcome these challenges with two technical developments: i) the use of perfluoroamines as carrier fluids, and ii) the use of Teflon capillaries for the formation, transport, and storage of plugs. We obtained high-quality crystals of membrane proteins and solved a crystal structure. This robust method requires inexpensive equipment and supplies, is accessible to most basic individual laboratories, and could find applications in a number of areas that require chemical, biochemical or biological screening and optimization.
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