Name: high-throughput protein expression generator using a microfluidic platform
Uploaded: 2012-08-23T12:00:00
Description: Watch this Scientific Journal Video about High-throughput Protein Expression Generator Using a Microfluidic Platform at JoVE.com

Dette arbejde blev støttet af Marie Curie international reintegrationsstipendium.

1. Device Fabrication <ol><li> Købt DTPA-D SU-8 kontrol skimmel og SPR220-7 flow mug fra Stanford Microfluidics Foundry ( <a href="http://www.stanford.edu/group/foundry" target="_blank">www.stanford.edu / group / støberi</a> ). </li><li> Udsætte silikoneforme til chlortrimethylsilan (TMCS) damp i 10 minutter til fremme af elastomer frigivelse efter bagetrin 9. </li><li> Der fremstilles en blanding af siliconebaseret elastomer og hærdningsmiddel (bland godt) i to forskellige forhold 5:01 og 20:1 for kont...

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	<li>Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+column:+Microfluidic+high-throughput+screening.">Integration column: Microfluidic high-throughput screening.</a> Integrative biology quantitative biosciences from nano to macro. 1, 19-29 (2009).</li><li>Hong, J. W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+nanoliter+systems.">Integrated nanoliter systems.</a> Nature. 21, 1179-1183 (2003).</li><li>Unger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Monolithic+Microfabricated+Valves+and+Pumps+by+Multilayer+Soft+Lithography.">A Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography.</a> Science. 288, 113-116 (2000).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nature. 6, 71-74 (2009).</li><li>Einav, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+hepatitis+C+target+and+its+pharmacological+inhibitors+by+microfluidic+affinity+analysis.">Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis.</a> Nature. 26, 1019-1027 (2008).</li><li>Fordyce, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+identification+and+biophysical+characterization+of+transcription-factor+binding+sites+with+microfluidic+affinity+analysis.">De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis.</a> Nature Biotechnology. 28, 962-967 (2010).</li><li>Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+activities+using+proteome+chips.">Global analysis of protein activities using proteome chips.</a> Science (New York, N.Y.). 293, 2101-2105 (2001).</li><li>Ramachandran, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+protein+microarrays.">Self-assembling protein microarrays.</a> Science (New York, N.Y.). 305, 86-90 (2004).</li><li>Zhong, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+processor+for+gene+expression+profiling+of+single+human+embryonic+stem+cells.">A microfluidic processor for gene expression profiling of single human embryonic stem cells.</a> Lab on a chip. 8, 68-74 (2008).</li><li>Kusnezow, W., Hoheisel, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+supports+for+microarray+immunoassays.">Solid supports for microarray immunoassays.</a> Journal of molecular recognition JMR. 16, 165-176 (2003).</li><li>Lundin, M., Monne, M., Widell, A., Von Heijne, G., Persson, M. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+of+the+membrane-associated+hepatitis+C+virus+protein+NS4B.">Topology of the membrane-associated hepatitis C virus protein NS4B.</a> Journal of virology. 77, 5428 (2003).</li></ol>

I denne afhandling præsenterer vi en metode til generation protein arrays i high-throughput ved hjælp af en mikrofluid platform. Array generation er baseret på microarray trykning af DNA-skabeloner og in vitro protein-ekspression fra DNA i mikrofluid enhed. Vores hidtil ukendte microfluidic platform har flere vigtige fordele i forhold til tiden anvendte metoder, som gør det lovende og generelt værktøj til proteomics. En fordel er med membranbundne proteiner. In vi...

<table border="1"><tbody><tr><td> Reagens / Udstyr </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> PDMS-Sylgard 184 </td><td> Dow Corning USA </td><td> ESSEX-DC </td></tr><tr><td> Chlortrimethylsilan (TMCS </td><td> Sigma-Aldrich </td><td> C72854 </td></tr><tr><td> Epoxy glasunderlag </td><td> CEL Associates USA </td><td> VEPO-25C </td></tr><tr><td> Polyethylen glycole (PEG) </td><td> Sigma-Aldrich </td><td> 81.260 </td></tr><tr><td> D-trehalose dihydrat </td><td> Sigma-Aldrich </td><td> T9531 </td></tr><tr><td> Biotinyleret-BSA </td><td> Pierce </td><td> PIR-29.130 </td></tr><tr><td> Neutravidin </td><td> Pierce </td><td> 31.050 </td></tr><tr><td> penta-His-biotin </td><td> Qiagen </td&gt; <td> 34.440 </td></tr><tr><td> Hepes </td><td> <a href="http://www.bioind.com/htmls/article.aspx?c0=741&amp;bsp=13384" target="_blank">Biological Industries</a> </td><td> 03-025-1B </td></tr><tr><td> TNT-T7 </td><td> Promega </td><td> L5540 </td></tr><tr><td> C-myc-antistof Cy3 </td><td> Sigma-Aldrich </td><td></td></tr><tr><td> Styreboks </td><td> Stanford Microfluidics Foundry </td><td></td></tr><tr><td> Mold </td><td> Stanford Microfluidics Foundry </td><td></td></tr><tr><td> Pin </td><td> New England små rør Corporation </td><td></td></tr><tr><td> Tygon microbore slange </td><td> Tygon </td><td> S-54-HL </td></tr><tr><td> Microarrayer </td><td> Bio Robotics </td><td> MicroGrid 610 </td></tr><tr><td> Silikone pins </td><td> Parallel Synafhandling </td><td> SMT-S75 </td></tr></tbody></table>

high-throughput protein expression generator using a microfluidic platform

Hastigt stigende områder, såsom systembiologi, kræver udviklingen og gennemførelsen af ​​nye teknologier, så high-throughput og high-fidelity målinger af store systemer. Microfluidics lover at opfylde mange af disse krav, såsom at udføre high-throughput screening eksperimenter på chippen, som omfatter biokemiske, biofysiske og cellebaserede assays 1. Eftersom de tidlige dage af mikrofluidik enheder, har dette felt drastisk udviklet, hvilket fører til udviklingen af mikrofluid stor skala integration 2,3. Denne teknologi giver mulighed for integration af tusindvis af mikromekaniske ventiler på en enkelt enhed med en porto-størrelse fodaftryk (figur 1). Vi har udviklet en high-throughput mikrofluid platform til frembringelse af in vitro-ekspression af protein arrays (fig. 2) navngivne PING (Protein Interaction Network Generator). Disse arrays kan tjene som en skabelon for mange eksperimentersåsom protein-protein-4, protein-RNA 5 eller protein-DNA 6 interaktioner. Anordningen består af tusinder af reaktionskamre, som er individuelt programmerede under anvendelse af en microarrayer. Justering af disse trykte microarrays til MicroFluidics enheder programmer hvert kammer med en enkelt plet eliminere potentiel kontaminering eller krydsreaktivitet Endvidere genererer microarrays under anvendelse af standard microarray spotting teknikker er også meget modulær, hvilket tillader opstillingsindretningen af proteiner 7, DNA 8, små molekyler, og endda kolloide suspensioner. De potentielle virkninger af mikrofluidik på biologiske videnskaber er betydelig. En række MicroFluidics baserede assays har allerede tilvejebragt hidtil ukendte indsigt i strukturen og funktionen af ​​biologiske systemer, og inden for mikrofluidik vil fortsætte med at påvirke biologi.

Watch this Scientific Journal Video about High-throughput Protein Expression Generator Using a Microfluidic Platform at JoVE.com

High-throughput Protein Expression Generator Using a Microfluidic Platform

Vi præsenterer en mikrofluid fremgangsmåde til ekspression af protein arrays. Anordningen består af tusinder af reaktionskamre kontrolleres af mikromekaniske ventiler. Den mikrofluid enhed er parret til en microarray-trykte genbibliotek. Disse gener er dernæst transkriberet og translateret on-chip, hvilket resulterer i et protein-array klar til eksperimentel anvendelse.

High-throughput Protein Expression Generator Ved hjælp af en Mikrofluid Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Research

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Bioengineering

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