Name: combining single-molecule manipulation and imaging for the study of protein-dna interactions
Uploaded: 2014-08-27T14:00:00
Description: Watch this Scientific Journal Video about Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions at JoVE.com

Vi tackar Gijs wuite, Erwin JG Peterman, och Peter Gross för hjälp med mikrofluidik och Alessia Tempestini för hjälp med provberedning. Denna forskning har finansierats av Europeiska unionens sjunde ramprogram (FP7 / 2007-2013) enligt bidragsavtal n ° 284.464 och från det italienska ministeriet för utbildning, universitet och forskning FIRB 2011 RBAP11X42L006, Futuro i Ricerca 2013 RBFR13V4M2, och inom ramen för Flagship Project Nanomax.

1 optisk pincett Setup med Nanometer Stabilitet Den experimentuppställning måste ge två optisk pincett med pekande stabilitet på nanometernivå och intensitet fluktuationer i fångst laser under 1%. Kombination av dessa villkor kommer att försäkra nanometer stabiliteten i hantel i typiska spänning (1 pN - några tiotal PN), fälla styvhet (0,1 pN / nm) och mätbandbredd (bild förvärv hastighet 20 sek -1). Ett system av experimentuppställningen visas i fig 1.</stron...

<ol>
	<li>Monico, C., Capitanio, M., Belcastro, G., Vanzi, F., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+to+Study+Protein-DNA+Interactions+in+Vitro+and+in+Living+Cells+at+the+Single-Molecule+Level.">Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level.</a> International journal of molecular sciences. 14, 3961-3992 (2013).</li><li>Tinoco, I., Gonzalez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+mechanisms,+one+molecule+at+a+time.">Biological mechanisms, one molecule at a time.</a> Genes &amp; development. 25, 1205-1231 (2011).</li><li>Capitanio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+molecular+motors+and+switches+at+the+single-molecule+level.">Exploring molecular motors and switches at the single-molecule level.</a> Micr. Res. Tech. 65, 194-204 (2004).</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>Block, S. M., Goldstein, L. S., Schnapp, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bead+movement+by+single+kinesin+molecules+studied+with+optical+tweezers.">Bead movement by single kinesin molecules studied with optical tweezers.</a> Nature. 348, 348-352 (1990).</li><li>Wang, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+velocity+measured+for+single+molecules+of+RNA+polymerase.">Force and velocity measured for single molecules of RNA polymerase.</a> Science. 282, 902-907 (1998).</li><li>Capitanio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+force-clamp+spectroscopy+of+single+molecules+reveals+load+dependence+of+myosin+working+stroke.">Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.</a> Nat Methods. 9, 1013-1019 (2012).</li><li>Thompson, R. E., Larson, D. R., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+nanometer+localization+analysis+for+individual+fluorescent+probes.">Precise nanometer localization analysis for individual fluorescent probes.</a> Biophys J. 82, 2775-2783 (2002).</li><li>Yildiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+V+walks+hand-over-hand:+single+fluorophore+imaging+with+1.5-nm+localization.">Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.</a> Science. 300, 2061-2065 (2003).</li><li>Biebricher, A., Wende, W., Escude, C., Pingoud, A., Desbiolles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+of+single+quantum+dot+labeled+EcoRV+sliding+along+DNA+manipulated+by+double+optical+tweezers.">Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers.</a> Biophys J. 96, 50-52 (2009).</li><li>Candelli, A., Wuite, G. J., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+optical+trapping,+fluorescence+microscopy+and+micro-fluidics+for+single+molecule+studies+of+DNA-protein+interactions.">Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions.</a> Physical chemistry chemical physics : PCCP. 13, 7263-7272 (2011).</li><li>Capitanio, M., Cicchi, R., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+and+time-shared+multiple+optical+tweezers+for+the+study+of+single+motor+proteins.">Continuous and time-shared multiple optical tweezers for the study of single motor proteins.</a> Optics and Lasers in Engineering. 45, 450-457 (2007).</li><li>Harada, Y., et al. <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+imaging+of+RNA+polymerase-DNA+interactions+in+real+time.">Single-molecule imaging of RNA polymerase-DNA interactions in real time.</a> Biophys J. 76, 709-715 (1999).</li><li>van Mameren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+RAD51+proteins+disassembling+from+nucleoprotein+filaments+under+tension.">Counting RAD51 proteins disassembling from nucleoprotein filaments under tension.</a> Nature. 457, 745-748 (2009).</li><li>Capitanio, M., Maggi, D., Vanzi, F., Pavone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiona+in+the+trap:+the+advantages+of+combining+optical+tweezers+and+fluorescence.">Fiona in the trap: the advantages of combining optical tweezers and fluorescence.</a> J Opt A: Pure Appl Opt. 9, s157 (2007).</li><li>Dijk, M. A., Kapitein, L. C., Mameren, J., Schmidt, C. F., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+optical+trapping+and+single-molecule+fluorescence+spectroscopy:+enhanced+photobleaching+of+fluorophores.">Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores.</a> J Phys Chem B. 108, 6479-6484 (2004).</li><li>Capitanio, M., Cicchi, R., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position+control+and+optical+manipulation+for+nanotechnology+applications.">Position control and optical manipulation for nanotechnology applications.</a> European Physical Journal B. 46, 1-8 (2005).</li><li>Capitanio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+optical+tweezers+with+differential+interference+contrast+signals.">Calibration of optical tweezers with differential interference contrast signals.</a> Review of Scientific Instruments. 73, 1687-1696 (2002).</li><li>Elangovan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+in+vitro+and+in+situ+study+of+kinetics+of+myosin+II+from+frog+skeletal+muscle.">An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle.</a> J Physiol. 590, 1227-1242 (2012).</li><li>Wuite, G. J. L., Davenport, R. J., Rappaport, A., 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=An+integrated+laser+trap/flow+control+video+microscope+for+the+study+of+single+biomolecules.">An integrated laser trap/flow control video microscope for the study of single biomolecules.</a> Biophysical Journal. 79, 1155-1167 (2000).</li><li>Brewer, L. R., Bianco, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+flow+cells+for+single-molecule+studies+of+DNA-protein+interactions.">Laminar flow cells for single-molecule studies of DNA-protein interactions.</a> Nat Methods. 5, 517-525 (2008).</li><li>Rutkauskas, D., Zhan, H. L., Matthews, K. S., Pavone, F. S., Vanzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetramer+opening+in+LacI-mediated+DNA+looping.">Tetramer opening in LacI-mediated DNA looping.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 16627-16632 (2009).</li><li>Marko, J. F., Siggia, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+DNA.">Stretching DNA.</a> Macromolecules. 28, 8759-8770 (1995).</li><li>van Mameren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+structure+of+DNA+during+overstretching+by+using+multicolor,+single-molecule+fluorescence+imaging.">Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.</a> Proc Natl Acad Sci U S A. 106, 18231-18236 (2009).</li><li>Wang, M. D., Yin, H., Landick, R., Gelles, J., 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=Stretching+DNA+with+optical+tweezers.">Stretching DNA with optical tweezers.</a> Biophys J. 72, 1335-1346 (1997).</li><li>Ma, H., Long, F., Zeng, S., Huang, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+precise+algorithm+based+on+maximum+radial+symmetry+for+single+molecule+localization.">Fast and precise algorithm based on maximum radial symmetry for single molecule localization.</a> Opt Lett. 37, 2481-2483 (2012).</li><li>Parthasarathy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+accurate+particle+tracking+by+calculation+of+radial+symmetry+centers.">Rapid, accurate particle tracking by calculation of radial symmetry centers.</a> Nat Methods. 9, 724-726 (2012).</li></ol>

Under det senaste decenniet har enstaka molekyl manipulation och avbildningstekniker sett stora framsteg i fråga om tidsmässiga och geografiska. Kombinationen av manipulation och avbildningstekniker är vid basen av kraftfulla instrument som nu går att kontrollera de mekaniska betingelserna för en enda biologisk polymer, såsom DNA, RNA eller cytoskelettala filament, och den samtidiga lokalisering av enstaka proteiner som interagerar med samma polymer . Styrning av de mekaniska förhållandena i instängd polymeren ...

Enda molekyl (SM) tekniker har utvecklats mycket under de senaste trettio åren för att svara på behovet att övervinna några av de begränsningar av traditionella, bulkmätningar lösnings 1-3. Den manipulation av enskilda biologiska molekyler har skapat möjligheten att mäta mekaniska egenskaper hos biopolymerer 4 och kontrollera de mekaniska parametrarna för protein-protein 5 och protein-DNA-interaktioner 6,7. SM fluorescensdetektion, å andra sidan, utgör en oerhört mångsidigt verktyg för att studera protein-aktivitet in vitro och in vivo, vilket leder till möjligheten att lokalisera och spåra enskilda molekyler med nanometerprecision. Genom montering av instrumentet punkt-spridningsfunktion till SM bild, i själva verket kan en utföra lokalisering med en precision beroende huvudsakligen på signal-till-brusförhållandet (SNR) och når en gräns av cirka en nanometer 8,9. Dessa metoder hitta kraftfullapplikationer i studiet av dynamiken i motorproteiner, liksom de diffusionsprocesser underliggande målet sökning i DNA-bindande proteiner. Förmågan att bestämma diffusionskonstanter som en funktion av DNA-sekvensen, uppehållstiden på målet och exakt mätning av DNA-längd utforskas under endimensionella diffusion händelser, representerar ett kraftfullt verktyg för att studera protein-DNA-interaktion dynamik och för undersökning av mekanismerna för specifikt mål ökning. Nyligen har kombinationen av dessa två tekniker produceras en ny generation av experimentella uppställningar 10-14 möjliggör samtidig manipulering av ett biologiskt substrat (t ex en aktin filament eller en DNA-molekyl) och detektion / lokalisering av en samverkande partner enzym (t.ex. myosin eller ett DNA-bindande protein). Fördelarna med dessa tekniker vilar i huvudsak på möjligheten att utöva mekanisk kontroll över den instängda polymeren, sålunda ENAbling studier av interaktionsdynamik kontra krafter eller moment. Dessutom tillåter den metod mätning av biokemiska reaktioner långt från ytan, undviker en av de viktigaste begränsningarna hos klassiska SM metoder, dvs behovet för immobilisering av molekyler under studien på en yta (glasskiva eller mikrosfärer). Kombinationen av två enkla molekyler tekniker kräver vinna flera tekniska svårigheter, främst till följd av kraven på mekanisk stabilitet och tillräcklig SNR (speciellt när det krävs lokalisering med nm precision) 15. Speciellt vid koppling SM fluorescensdetektion med optisk pincett, minskning av buller och fotoblekning från fångstinfraröda lasrar 16 och kontroll av biokemiska buffertar för montering av de biologiska komplex och prestanda för experimentella mätningar 11 är av största vikt. Här beskriver vi de metoder för framgångsriktmätningar i en dubbel fångst / SM Fluorescens lokaliseringsinställningar. Metodiken illustreras med exempel på laktosrepressorproteinet (LacI) fluorescerande (med Atto532) och detekteras som den binder till en DNA-molekyl (fångade mellan två optisk pincett) innehåller specifika Laci bindande sekvenser (dvs operatörer). Vi visar effektiviteten av metoden i detektering av bindning av Lacl till DNA och diffusion utmed sin kontur i mål-sökningsprocessen. Metoden kan tillämpas på alla kombinationer av DNA-sekvensen och DNA-bindande protein, liksom till andra system (mikrotubuli eller aktinfilament och motorproteiner som interagerar med dem).

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:203px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td style="width:151px;">Comments/Description</td>
			<td style="width:203px;">Web Address</td>
		</tr>
		<tr height="114">
			<td height="114" style="height:115px;">elastomeric isolators</td>
			<td style="width:141px;">Newport</td>
			<td style="width:167px;">Newdamp</td>
			<td style="width:151px;">Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies</td>
			<td><a href="http://www.newport.com/">http://www.newport.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical isolator</td>
			<td style="width:141px;">Optics for Research</td>
			<td style="width:167px;">IO-3-YAG-VHP</td>
			<td></td>
			<td><a href="http://www.ofr.com/">http://www.ofr.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nd:YAG laser, 1064 nm wavelength</td>
			<td style="width:141px;">Spectra-Physics</td>
			<td style="width:167px;">Millennia IR</td>
			<td></td>
			<td><a href="http://www.newport.com/">http://www.newport.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acousto-optic deflectors (AODs)</td>
			<td style="width:141px;">A&#38;A optoelectronic</td>
			<td style="width:167px;">DTS-XY 250</td>
			<td></td>
			<td><a href="http://www.aaoptoelectronic.com/">http://www.aaoptoelectronic.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Direct Digital Synthesizers</td>
			<td style="width:141px;">Analog Devices</td>
			<td style="width:167px;"></td>
			<td></td>
			<td><a href="http://www.analog.com/">http://www.analog.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quadrant detector photodiodes</td>
			<td style="width:141px;">OSI optoelectronics</td>
			<td style="width:167px;">SPOT-15-YAG</td>
			<td></td>
			<td><a href="http://www.osioptoelectronics.com/">http://www.osioptoelectronics.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">DIO and FPGA board</td>
			<td style="width:141px;">National Instruments</td>
			<td style="width:167px;">NI-PCI-7830R</td>
			<td></td>
			<td><a href="http://www.ni.com/">http://www.ni.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Halogen lamp</td>
			<td style="width:141px;">Schott</td>
			<td style="width:167px;">KL 1500 LCD</td>
			<td></td>
			<td><a href="http://www.schott.com/">http://www.schott.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Condenser</td>
			<td style="width:141px;">Olympus</td>
			<td style="width:167px;">U-AAC 1.4NA Aplanat Apchromat</td>
			<td></td>
			<td><a href="http://www.olympus-global.com/en/">http://www.olympus-global.com/en/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Objective</td>
			<td style="width:141px;">Nikon</td>
			<td style="width:167px;">CFI Plan Apochromat 60x 1.2NA water immersion</td>
			<td></td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">532 nm laser</td>
			<td style="width:141px;">Coherent</td>
			<td style="width:167px;">Sapphire</td>
			<td></td>
			<td><a href="http://www.coherent.com/">http://www.coherent.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CCD 200X and 2000X</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">XC-ST70 CE</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">electron-multiplied CCD</td>
			<td style="width:141px;">Hamamatsu&#160;</td>
			<td style="width:167px;">C9100-13</td>
			<td></td>
			<td><a href="http://www.hamamatsu.com/">http://www.hamamatsu.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">piezo stage with nm-accuracy</td>
			<td style="width:141px;">Physik Instrumente</td>
			<td style="width:167px;">P-527.2CL&#160;</td>
			<td></td>
			<td><a href="http://www.physikinstrumente.com/">http://www.physikinstrumente.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Emission Filter</td>
			<td style="width:141px;">Chroma Technologies</td>
			<td style="width:167px;">600/100m</td>
			<td></td>
			<td><a href="http://www.chroma.com/">http://www.chroma.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silica beads (1.54 mm)</td>
			<td style="width:141px;">Bangs Laboratories</td>
			<td style="width:167px;">SS04N/5303</td>
			<td></td>
			<td><a href="http://www.bangslabs.com/">http://www.bangslabs.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum (BSA)</td>
			<td style="width:141px;">Sigma Aldrich</td>
			<td style="width:167px;">B4287</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pentyl acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">46022</td>
			<td style="width:151px;">Flammable liquid and vapour (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">nitrocellulose</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">N8267-5EA</td>
			<td style="width:151px;">Flammable solid&#160; (No 1272/2008)</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">heat block</td>
			<td style="width:141px;">MPM Instruments Srl</td>
			<td style="width:167px;">M502-HBD</td>
			<td style="width:151px;">with 2 removable blocks; preheated at 120&#176; C</td>
			<td><a href="http://www.mpminstruments.com/">http://www.mpminstruments.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoPort assemblies</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">N-333</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">polyetheretherketone tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific Inc.</td>
			<td style="width:167px;">1535</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass</td>
			<td style="width:141px;"></td>
			<td style="width:167px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luer lock-tip syringes 2.5 mL</td>
			<td style="width:141px;">Terumo</td>
			<td style="width:167px;">SS 02LZ1</td>
			<td></td>
			<td><a href="http://www.terumomedical.com/">http://www.terumomedical.com</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">shut-off valves&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">P-732</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">flangeless fittings&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">LT-111</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">fluorinated ethylene propylene tubing&#160;</td>
			<td style="width:141px;">Upchurch Scientific, Inc.</td>
			<td style="width:167px;">1549</td>
			<td></td>
			<td><a href="http://www.upchurch.com/">http://www.upchurch.com/</a></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:203px;">two computer-controlled solenoid valves</td>
			<td style="width:141px;">Clippard, Cincinnati, USA</td>
			<td style="width:167px;">ET-2-H-M5</td>
			<td></td>
			<td><a href="http://www.clippard.com/">http://www.clippard.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pressure transducer</td>
			<td style="width:141px;">Druck LTD</td>
			<td style="width:167px;">PTX 1400</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biotin-14-dCTP&#160;</td>
			<td style="width:141px;">Life Technologies</td>
			<td style="width:167px;">19518-018</td>
			<td></td>
			<td><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Terminal deoxynucleotidyl Transferase (TdT)</td>
			<td style="width:141px;">Thermoscientific</td>
			<td style="width:167px;">EP0161</td>
			<td></td>
			<td><a href="http://www.thermoscientificbio.com/">http://www.thermoscientificbio.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATTO532 maleimide</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">68499</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="157">
			<td height="157" style="height:157px;width:203px;">N,N-dimethylformamide (DMF)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">227056</td>
			<td style="width:151px;">Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP)&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">C4706</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">L-Glutathione reduced (GSH)</td>
			<td>Sigma Aldrich</td>
			<td style="width:167px;">G4251</td>
			<td style="width:151px;">Acute toxicity, Oral (Category 5), H303</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com/</a></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10&#160;kDa cutoff spin concentrators</td>
			<td style="width:141px;">Merck Millipore</td>
			<td style="width:167px;">UFC901024</td>
			<td></td>
			<td><a href="http://www.merckmillipore.it/">http://www.merckmillipore.it/</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">streptavidin-coated polystyrene beads 1,87 &#181;m</td>
			<td style="width:141px;">Spherotech, Inc.</td>
			<td style="width:167px;">SVP-15-5</td>
			<td></td>
			<td><a href="http://www.spherotech.com/">http://www.spherotech.com/</a></td>
		</tr>
	</tbody>
</table>

combining single-molecule manipulation and imaging for the study of protein-dna interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

I ett lyckat experiment, genomgår en (eller flera) märkta proteiner bindande / ej bindande och / eller monodimensional diffusion längs DNA-molekylen (Figur 3A). Lokalisering av proteiner längs DNA-molekylen möjliggör kvantifiering av kinetiska parametrar som en funktion av DNA-sekvensen. När buffertförhållanden orsakar 1D diffusion tillämpas, är det möjligt att följa protein banor, och bestämmer till exempel diffusionskoefficienten D 1D. Den exakta p...

Watch this Scientific Journal Video about Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions at JoVE.com

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Kombinera enda molekyl manipulation och Imaging för studier av protein-DNA-interaktioner

Här beskriver vi instrumentering och metoder för att detektera enstaka fluorescensmärkta proteinmolekyler som interagerar med en enda DNA-molekyl upphängd mellan två optiskt fångade mikrosfärer.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testa Visual Känsligheten för hastighet och rörelseriktning i Ödlor

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Datorgenererade djurmodell Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Enda molekyl Metoder för att följa förändringar i tvåskiktsmembran elastiska egenskaper

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Isolering och genetisk manipulation av vuxen Cardiac myocyter för Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single-Molecule Imaging of Nuclear Transport

Enda molekyl avbildning av kärntransport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Enda molekyl avbildning av genreglering<em&gt; In vivo</em&gt; Använda Cotranslational Aktivering genom klyvning (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Spirande jästprotein extraktion och rening för studien av funktion, interaktioner, och post-translationella modifieringar

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Kromatin Immunoprecipitation analys för identifiering av Arabidopsis Protein-DNA-interaktioner<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chipet-exo Metod: Identifiera protein-DNA interaktioner med nära baspar Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Study of Protein-protein Interactions in Autophagy Research

Studie av Protein-protein interaktioner i autofagi forskning

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...