Name: multiplexed single-molecule force proteolysis measurements using magnetic tweezers
Uploaded: 2012-07-25T11:00:00
Description: Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Dit werk werd ondersteund door de Burroughs Wellcome Career Award op het Wetenschappelijk Interface (ARD), de National Institutes of Health door New Innovator Award van de NIH Director&#39;s Programma 1-DP2-OD007078 (ARD), de William Bowes Jr Stanford Graduate Fellowship (ASA ), en aan de Stanford Cardiovascular Institute Jongere Predoctoraal Fellowship (JC). De auteurs danken James Spudich voor lenen microscopie apparatuur.

1. Flow Cell Bereiding <ol><li> Dekglaasjes (# 1.5, 22x22 mm en 22x40 mm, VWR) worden gereinigd met ultrasoonapparaat. <ol><li> Voeg de dekglaasjes tot een klein glazen fles kunnen houden dekglaasjes en passend in de ultrasoonapparaat (zie stap 2). </li><li> Vul de container met isopropanol en ultrasone trillingen in een bad ultrasoonapparaat gedurende 20 minuten. </li><li> Gooi de isopropanol en spoel de dekglaasjes met een ruime hoeveelheid gedemineraliseerd water geproduceerd door een Barnsted MilliQ apparaat of so...

<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>

Dit protocol beschrijft een nieuwe toepassing voor een klassieke single molecule techniek. Magnetisch pincet laten medium tot high-throughput enkel molecuul testen op een kosten-efficiënte manier. Echter, net als alle andere experimentele technieken zijn er uitdagingen en mogelijke valkuilen. Beperkingen van magnetische pincet In vergelijking met een optische val van de ruimtelijke en temporele resolutie van een MT apparaat is laag. Bovendien is de krachten opgewek...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogus nummer </td></tr><tr><td> Micro Dekglas # 1.5 (22x22) </td><td> VWR </td><td> 48366-067 </td></tr><tr><td> Micro Dekglas # 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> Microcontroller Ultracel YM-100 </td><td> Millipore </td><td> 42413 </td></tr><tr><td> Anti-Digoxigenine </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&gt; <tr><td> Anti-myc antilichaam </td><td> Invitrogen </td><td> 46-0603 </td></tr><tr><td> Bovine serum albumine </td><td> Sigma </td><td> B4287-5G </td></tr><tr><td> Dynabeads M-280 Streptavidine </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> Dynabeads MyOne T1 Streptavidine </td><td> Invitrogen </td><td> 658.01D </td></tr><tr><td> p-acetaat Aminophenylmercuric </td><td> Calbiochem </td><td> 164610 </td></tr><tr><td> Biotine-maleïmide </td><td> Sigma Aldrich </td><td> B1267 </td></tr><tr><td> Biotine gelabelde oligo </td><td> IDT DNA </td><td> Synthese op maat </td></tr><tr><td> Digoxigenine gelabelde oligo </td><td> IDT DNA </td><td> Synthese op maat </td></tr><tr><td> Collageenpeptide gen </td><td> DNA-2.0 </td><td> Synthese op maat </td></tr><tr><td> MMP-1 cDNA </td><td> Harvard Plasmide Database </td><td></td></tr><tr><td> z-translator </td><td> Thorlabs </td><td> MTS50 </td></tr><tr><td> Servo-controller voor vertaler </td><td> Thorlabs </td><td> TDC001 </td></tr></tbody></table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

Het genereren en detectie van mechanische krachten is een alomtegenwoordig aspect van de cel fysiologie, met directe relevantie voor kanker metastase 1, 2 en atherogenese wondgenezing 3. In elk van deze voorbeelden cellen zowel uitoefenen van kracht op de omgeving en tegelijkertijd enzymatisch vorm van de extracellulaire matrix (ECM). Het effect van krachten op ECM is zo uitgegroeid tot een gebied van groot belang vanwege de mogelijke biologische en medische belang 4-7. Enkel molecuul technieken zoals optische trapping 8, atomic force microscopie 9, en magnetische pincet 10,11 kunnen de onderzoekers om de functie van enzymen op moleculair niveau door het uitoefenen van krachten op afzonderlijke eiwitten te onderzoeken. Van deze technieken, magnetische pincet (MT) zijn opmerkelijk vanwege hun lage kosten en hoge doorvoer. MT oefenen krachten in het bereik van 1-100 ~ pN en kunnen milliseconde temporele resolutiekwaliteiten die goed zijn voor de studie van enzym-mechanisme afgestemd op de single-molecule level 12. Hier rapporteren een zeer parallelizable MT assay het effect van kracht op de Proteolyse van een eiwitmoleculen bestuderen. Wij geven het specifieke voorbeeld van de Proteolyse van een trimere collageen peptide matrix metalloproteinase 1 (MMP-1), maar deze assay kan gemakkelijk worden aangepast aan andere substraten en proteasen bestuderen.

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 dit artikel beschrijven we het gebruik van magnetische pincet om het effect van geweld op enzymatische proteolyse studeren aan de enkel molecuul niveau in een zeer parallelizable manier.

Multiplexed Single-molecule force Proteolyse metingen met behulp van magnetisch pincet

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

Research

JoVE Journal

Bioengineering

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Stretching korte sequenties van DNA met Constant Force Axiale optisch pincet

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Buigstijfheid Metingen van Biopolymeren gebruik Zweefvliegen Assays

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Onderzoeken Receptor-ligand systemen van de Cellulosome met AFM-gebaseerde Single-molecule Force Spectroscopie

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Magnetic Tweezers for the Measurement of Twist and Torque

Magnetische pincet voor het meten van Twist en Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Hoge-resolutie Spatiotemporal Analyse van Receptor Dynamics door Single-molecule fluorescentie microscopie

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Single Molecule Fluorescentie Microscopie op Ondersteunde Planar dubbellagen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Label-free Single Molecule Detectie Met behulp microtoroıde optische resonatoren

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Rapid Scan Electron Paramagnetische Resonantie opent nieuwe wegen voor Imaging Fysiologisch belangrijke parameters<em&gt; In Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Een gids voor het bouwen van een sterk geneigd geveegd tegel Microscoop voor uitgebreide veld-of-view Single-molecuul Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Karakteriseren single-molecule conformatieveranderingen onder schuifstroom met fluorescentiemicroscopie

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...