Multiparameter screening on SlipChip used for nanoliter protein crystallization combining free interface diffusion and microbatch methods.
Multiparameter screening on SlipChip used for nanoliter protein crystallization combining free interface diffusion and microbatch methods.
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DOI：
10.1021/ja908558m
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发表时间：
2010-01-13
影响因子：
15
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
本文介绍了两种基于SlipChip的蛋白质结晶方法：一种基于SlipChip的自由界面扩散（FID）方法和一种基于SlipChip的复合方法，该方法在单个设备中同时执行微批次和FID结晶方法。FID滑动芯片被设计为筛选多种试剂，每种在多个扩散平衡时间，并且通过筛选两种蛋白质（来自结核分枝杆菌的烯酰-CoA水合酶和来自牛巴氏杆菌的二氢叶酸还原酶/胸苷酸合酶）在各自5个不同平衡时间针对48种不同试剂的结晶条件来验证，消耗12 μL每种蛋白质，使用三个滑动芯片进行总共480次实验。复合滑动芯片被设计为筛选多种试剂，每种试剂在多个混合比和多个平衡时间，并通过筛选两种蛋白质的结晶条件进行验证，所述两种蛋白质是来自结核分枝杆菌的烯酰辅酶A水合酶和来自牛巴氏杆菌的二氢叶酸还原酶/胸苷酸合酶。为了防止交叉污染，同时保持颈部通道中的溶液对于FID稳定，滑动芯片的板被蚀刻有微孔图案。该纳米图案用于增加水溶液在硅烷化玻璃表面上的接触角。复合滑动芯片增加了成功结晶条件的数量，并确定了比单独的FID和微批筛选更多的结晶条件。使用在滑动芯片筛选期间鉴定的条件在孔板中按比例放大结晶实验，并且获得X射线衍射数据，以在1.95 μ m分辨率下产生二氢叶酸还原酶/胸苷酸合酶的蛋白质结构。这种自由界面扩散方法提供了在微流体装置中设置梯度的方便且高通量的方法，并且可以在基于细胞的测定中找到另外的应用。
This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis against 48 different reagents at 5 different equilibration times each, consuming 12 μL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 Å resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices, and may find additional applications in cell-based assays.
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影响因子：
15
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.
DOI：
10.1107/s0907444908028060
发表时间：
2008-11
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Gerdts CJ;Elliott M;Lovell S;Mixon MB;Napuli AJ;Staker BL;Nollert P;Stewart L
通讯作者：
Stewart L
影响因子：
56.9
作者：
Unger, MA;Chou, HP;Quake, SR
通讯作者：
Quake, SR
DOI：
10.1107/s0907444993014350
发表时间：
1994-07-01
影响因子：
2.2
作者：
GARCIARUIZ, JM;MORENO, A
通讯作者：
MORENO, A
影响因子：
4
作者：
Wu, Liang;Li, Guann-Pyng;Bachman, Mark
通讯作者：
Bachman, Mark