This work was supported by the Burroughs Wellcome Career Award at the Scientific Interface (A.R.D.), the National Institutes of Health through the NIH Director's New Innovator Award Program 1-DP2-OD007078 (A.R.D.), the William Bowes Jr. Stanford Graduate Fellowship (A.S.A.), and the Stanford Cardiovascular Institute Younger Predoctoral Fellowship (J.C.). The authors thank James Spudich for loaning microscopy equipment.

1. Flow Cell Preparation
<ol>
	<li>Coverslips (#1.5, 22x22 mm and 22x40 mm, VWR) are cleaned using sonication.
	<ol>
		<li>Add the coverslips to a small glass container capable of holding coverslips and fitting in the sonicator (see step 2).</li>
		<li>Fill the container with isopropanol and sonicate in a bath sonicator for 20 minutes.</li>
		<li>Discard the isopropanol and rinse the coverslips with copious quantities of deionized water produced by a Barnsted MilliQ apparatus or similar device. Fill the container with ...

<ol>
	<li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+cancer+be+reversed+by+engineering+the+tumor+microenvironment.">Can cancer be reversed by engineering the tumor microenvironment.</a> Semin. Cancer Biol. 18, 356-364 (2008).</li><li>Hahn, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+vascular+physiology+and+atherogenesis.">Mechanotransduction in vascular physiology and atherogenesis.</a> Nat. Rev. Mol. Cell Biol. 10, 53-62 (2009).</li><li>Laurens, N., Koolwijk, P., de Maat, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+structure+and+wound+healing.">Fibrin structure and wound healing.</a> J. Thromb. Haemost. 4, 932-939 (2006).</li><li>Adhikari, A. S., Chai, J., Dunn, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Load+Induces+a+100-Fold+Increase+in+the+Rate+of+Collagen+Proteolysis+by+MMP-1.">Mechanical Load Induces a 100-Fold Increase in the Rate of Collagen Proteolysis by MMP-1.</a> J. Am. Chem. Soc. 133, (2011).</li><li>Zareian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+collagen/enzyme+mechanochemistry+in+native+tissue+with+dynamic,+enzyme-induced+creep.">Probing collagen/enzyme mechanochemistry in native tissue with dynamic, enzyme-induced creep.</a> Langmuir. 26, (2010).</li><li>Ellsmere, J. C., Khanna, R. A., Lee, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+loading+of+bovine+pericardium+accelerates+enzymatic+degradation.">Mechanical loading of bovine pericardium accelerates enzymatic degradation.</a> Biomaterials. 20, 1143-1150 (1999).</li><li>Jesudason, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+regulate+elastase+activity+and+binding+site+availability+in+lung+elastin.">Mechanical forces regulate elastase activity and binding site availability in lung elastin.</a> Biophys. J. 99, 3076-3083 (2010).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev. Sci. Instrum. 75, 2787-2809 (2004).</li><li>Lee, C. K., Wang, Y. M., Huang, L. S., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy:+determination+of+unbinding+force,+off+rate+and+energy+barrier+for+protein-ligand+interaction.">Atomic force microscopy: determination of unbinding force, off rate and energy barrier for protein-ligand interaction.</a> Micron. 38, 446-461 (2007).</li><li>Smith, S. B., Finzi, L., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+mechanical+measurements+of+the+elasticity+of+single+DNA+molecules+by+using+magnetic+beads.">Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads.</a> Science. 258, 1122-1126 (1992).</li><li>Tanase, M., Biais, N., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers+in+cell+biology.">Magnetic tweezers in cell biology.</a> Methods Cell Biol. 83, 473-493 (2007).</li><li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nat. Methods. 5, 491-505 (2008).</li><li>Frank, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+short+collagen-like+triple+helices+by+protein+engineering.">Stabilization of short collagen-like triple helices by protein engineering.</a> J. Mol. Biol. 308, 1081-1089 (2001).</li><li>Stetefeld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+stabilization+at+atomic+level:+crystal+structure+of+designed+(GlyProPro)10foldon.">Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon.</a> Structure. 11, 339-346 (2003).</li><li>Chung, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+unwinds+triple-helical+collagen+prior+to+peptide+bond+hydrolysis.">Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis.</a> EMBO J. 23, 3020-3030 (2004).</li><li>Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+the+specific+adhesion+of+cells+to+cells.">Models for the specific adhesion of cells to cells.</a> Science. 200, 618-627 (1978).</li><li>Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-molecule techniques: a laboratory manual. , (2008).</li><li>Graneli, A., Yeykal, C. C., Prasad, T. K., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+arrays+of+individual+DNA+molecules+tethered+to+supported+lipid+bilayers.">Organized arrays of individual DNA molecules tethered to supported lipid bilayers.</a> Langmuir. 22, 292-299 (2006).</li><li>Danilowicz, C., Greenfield, D., Prentiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+ligand-receptor+complexes+using+magnetic+tweezers.">Dissociation of ligand-receptor complexes using magnetic tweezers.</a> Anal. Chem. 77, 3023-3028 (2005).</li><li>Zhang, X., Halvorsen, K., Zhang, C. Z., Wong, W. P., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoenzymatic+cleavage+of+the+ultralarge+vascular+protein+von+Willebrand+factor.">Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor.</a> Science. 324, 1330-1334 (2009).</li><li>Woodside, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 6190-6195 (2006).</li><li>del Rio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+single+talin+rod+molecules+activates+vinculin+binding.">Stretching single talin rod molecules activates vinculin binding.</a> Science. 323, 638-641 (2009).</li><li>Gosse, C., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers:+micromanipulation+and+force+measurement+at+the+molecular+level.">Magnetic tweezers: micromanipulation and force measurement at the molecular level.</a> Biophys. J. 82, 3314-3329 (2002).</li></ol>

This protocol describes a new use for a classical single molecule technique. Magnetic tweezers allow medium to high-throughput single molecule assays in a cost-efficient manner. However, like all experimental techniques there are challenges and potential pitfalls.
Limitations of magnetic tweezers
Compared to an optical trap the spatial and temporal resolution of a MT apparatus is low. Moreover, the forces generated by the simple MT described here are 30 pN or less, sig...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Micro Cover Glass #1.5 (22x22)</td>
 <td>VWR</td>
 <td>48366-067</td>
 </tr>
 <tr>
 <td>Micro Cover Glass #1.5 (22x40)</td>
 <td>VWR</td>
 <td>48393-048</td>
 </tr>
 <tr>
 <td>Lambda DNA</td>
 <td>Invitrogen</td>
 <td>25250-010</td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>Invitrogen</td>
 <td>15224-041</td>
 </tr>
 <tr>
 <td>Microcon Ultracel YM-100</td>
 <td>Millipore</td>
 <td>42413</td>
 </tr>
 <tr>
 <td>Anti-Digoxigenin</td>
 <td>Roche Diagnostics</td>
 <td>11-333-089-001</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P9416-100ML</td>
 </tr>
 <tr>
 <td>Anti-myc Antibody</td>
 <td>Invitrogen</td>
 <td>46-0603</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>B4287-5G</td>
 </tr>
 <tr>
 <td>Dynabeads M-280 Streptavidin</td>
 <td>Invitrogen</td>
 <td>658.01D</td>
 </tr>
 <tr>
 <td>Dynabeads MyOne T1 Streptavidin</td>
 <td>Invitrogen</td>
 <td>658.01D</td>
 </tr>
 <tr>
 <td>p-Aminophenylmercuric Acetate</td>
 <td>Calbiochem</td>
 <td>164610</td>
 </tr>
 <tr>
 <td>Biotin-Maleimide</td>
 <td>Sigma Aldrich</td>
 <td>B1267</td>
 </tr>
 <tr>
 <td>Biotin labeled oligo</td>
 <td>IDT DNA</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>Digoxigenin labeled oligo</td>
 <td>IDT DNA</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>Collagen peptide gene</td>
 <td>DNA 2.0</td>
 <td>Custom synthesis</td>
 </tr>
 <tr>
 <td>MMP-1 cDNA</td>
 <td>Harvard Plasmid Database</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>z-translator</td>
 <td>Thorlabs</td>
 <td>MTS50</td>
 </tr>
 <tr>
 <td>Servo controller for translator</td>
 <td>Thorlabs</td>
 <td>TDC001</td>
 </tr>
 </tbody>
 </table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

multiplexed-single-molecule-force-proteolysis-measurements-using

Research

JoVE Journal

Bioengineering

11.5K 조회수.  Stanford University. 이 문서에서 우리는 높은 parallelizable 방식으로 단일 분자 수준에서 효소 proteolysis에 대한 힘의 효과를 공부하는 자기 족집게의 사용을 설명합니다.