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

This work was supported by Marie Curie international reintegration grant.

1. Device Fabrication 
<ol>
 <li>Purchased DTPA-D SU-8 control mold and SPR220-7 flow mold from the Stanford Microfluidics Foundry (<a href="http://www.stanford.edu/group/foundry" target="_blank">www.stanford.edu/group/foundry</a>).</li>
 <li>Expose the silicone molds to chlorotrimethylsilane (TMCS) vapor for 10 min to promote elastomer release after the baking steps9. </li>
 <li>Prepare a mixture of silicone based elastomer and curing agent (mix well) in two different ratios 5:1 and 20:1 for the contro...

<ol>
	<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>

In this paper we present a method for generation protein arrays in high-throughput using a microfluidic platform. The array generation is based on microarray printing of DNA templates and in vitro protein expression from the DNA within the microfluidic device. 
Our novel microfluidic platform has several important advantages over currently used methods, which make it a promising and general tool for proteomics. One advantage is with membrane-bound proteins. In vitro protein s...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>PDMS- SYLGARD 184</td>
 <td>Dow Corning USA</td>
 <td>ESSEX-DC</td>
 </tr>
 <tr>
 <td>Chlorotrimethylsilane (TMCS </td>
 <td>Sigma-Aldrich </td>
 <td>C72854 </td>
 </tr>
 <tr>
 <td>Epoxy coated glass substrates </td>
 <td>CEL Associates USA</td>
 <td>VEPO-25C</td>
 </tr>
 <tr>
 <td>Poly ethylene glycole (PEG)</td>
 <td>Sigma-Aldrich</td>
 <td>81260</td>
 </tr>
 <tr>
 <td>D-trehalose dihydrate</td>
 <td>Sigma-Aldrich</td>
 <td>T9531</td>
 </tr>
 <tr>
 <td>Biotinylated-BSA</td>
 <td>Pierce </td>
 <td>PIR-29130</td>
 </tr>
 <tr>
 <td>Neutravidin </td>
 <td>Pierce </td>
 <td>31050</td>
 </tr>
 <tr>
 <td>penta-His-biotin</td>
 <td>Qiagen</td>
 <td>34440</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 Cy3 antibody</td>
 <td>Sigma -Aldrich</td>
 <td></td>
 </tr>
 <tr>
 <td>Control box</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 Small Tubes Corporation</td>
 <td></td>
 </tr>
 <tr>
 <td>Tygon microbore tubing</td>
 <td>Tygon</td>
 <td>S-54-HL</td>
 </tr>
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 <td>Microarrayer</td>
 <td>Bio Robotics</td>
 <td>MicroGrid 610</td>
 </tr>
 <tr>
 <td>Silicone pins</td>
 <td>Parallel Synthesis</td>
 <td>SMT-S75</td>
 </tr>
 </tbody>
</table>

high-throughput protein expression generator using a microfluidic platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

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

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

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

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