Multiparameter Screening on SlipChip Used for Nanoliter Protein Crystallization Combining Free Interface Diffusion and Microbatch Methods.
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 scr...
| Publicado en: | Journal of the American Chemical Society Vol. 132; no. 1; pp. 112 - 120 |
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| Autores principales: | , , |
| Formato: | Artículo |
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American Chemical Society
1/13/2010
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| Materias: | |
| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=48230200&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 48230200 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 1/13/2010 vid: 132 iid: 1 pid: 997 pub: American Chemical Society artinfo: ui: 48230200 10.1021/ja908558m ppf: 112 ppct: 8 formats: tig: atl: Multiparameter Screening on SlipChip Used for Nanoliter Protein Crystallization Combining Free Interface Diffusion and Microbatch Methods. aug: au: Liang Li Wenbin Du Ismagilov, Rustern F. affil: Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637 su: Crystallization Protein synthesis Diffusion processes Mycobacterium tuberculosis X-ray diffraction Microfluidic devices sug: subj: Crystallization Protein synthesis Diffusion processes Mycobacterium tuberculosis X-ray diffraction Microfluidic devices ab: 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 five 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. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2010 holdings: @attributes: islocal: N |
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