This work was supported in part by a National Science Foundation grant DMS-1463234, National Institutes of Health grants HL082808 and AI133634, and Lehigh University internal funding.

1. Preparing VWF
<ol>
	<li>Reconstitute human plasma VWF to prepare it for the labeling reactions. Add 100 &#181;L of deionized (DI) water to 100 &#181;g of lyophilized VWF to create a 1 mg/mL VWF stock solution.</li>
	<li>Dialyze VWF stock solution in order to remove excess glycine, thereby increasing the biotin and fluorophore labeling efficiency.
	<ol>
		<li>Transfer 50 &#181;L of VWF stock solution into a 0.1 mL dialysis unit with a 10,000 molecular weight cut-off and seal with a cap. Store the remaining stock solu...

<ol>
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P., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-polymer+dynamics+in+steady+shear+flow.">Single-polymer dynamics in steady shear flow.</a> Science. 283 (5408), 1724-1727 (1999).</li><li>LeDuc, P., Haber, C., Bao, G., Writz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+individual+flexible+polymers+in+a+shear+flow.">Dynamics of individual flexible polymers in a shear flow.</a> Nature. 399 (6736), 564-566 (1999).</li><li>Huber, B., Harasim, M., Wunderlich, B., Kr&#246;ger, M., Bausch, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor,+Jedi+knight+of+the+bloodstream.">von Willebrand factor, Jedi knight of the bloodstream.</a> Blood. 124 (9), 1412-1425 (2014).</li><li>Merchant, K. A., Best, R. B., Louis, J. M., Gopich, I. V., Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+unfolded+states+of+proteins+using+single-molecule+FRET+spectroscopy+and+molecular+simulations.">Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (5), 1528-1533 (2007).</li><li>Neuman, K. 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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=Shear-dependent+changes+in+the+three-dimensional+structure+of+human+von+Willebrand+factor.">Shear-dependent changes in the three-dimensional structure of human von Willebrand factor.</a> Blood. 88 (8), 2939-2950 (1996).</li><li>Roiter, Y., Minko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM+single+molecule+experiments+at+the+solid-liquid+interface:+In+situ+conformation+of+adsorbed+flexible+polyelectrolyte+chains.">AFM single molecule experiments at the solid-liquid interface: In situ conformation of adsorbed flexible polyelectrolyte chains.</a> Journal of the American Chemical Society. 127 (45), 15688-15689 (2005).</li><li>Aznauryan, 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=Comprehensive+structural+and+dynamical+view+of+an+unfolded+protein+from+the+combination+of+single-molecule+FRET,+NMR,+and+SAXS.">Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (37), 5389-5398 (2016).</li><li>Schuler, B., Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+studied+by+single-molecule+FRET.">Protein folding studied by single-molecule FRET.</a> Current Opinion in Structural Biology. 18 (1), 16-26 (2008).</li><li>Howorka, S., Siwy, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore+analytics:+sensing+of+single+molecules.">Nanopore analytics: sensing of single molecules.</a> Chemical Society Reviews. 38 (8), 2360-2384 (2008).</li><li>Talaga, D. S., Li, J. <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+protein+unfolding+in+solid+state+nanopores.">Single-molecule protein unfolding in solid state nanopores.</a> Journal of the American Chemical Society. 131 (26), 9287-9297 (2009).</li><li>Moerner, W. E., Fromm, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+single-molecule+fluorescence+spectroscopy+and+microscopy.">Methods of single-molecule fluorescence spectroscopy and microscopy.</a> Review of Scientific Instruments. 74 (8), 3597-3619 (2003).</li><li>Xia, T., Li, N., Fang, X. H. <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+Fluorescence+Imaging+in+Living+Cells.">Single-Molecule Fluorescence Imaging in Living Cells.</a> Annual Review of Physical Chemistry. 64, 459-480 (2013).</li><li>Fu, H., 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=Flow-induced+elongation+of+von+Willebrand+factor+precedes+tension-dependent+activation.">Flow-induced elongation of von Willebrand factor precedes tension-dependent activation.</a> Nature Communications. 8 (324), 1-12 (2017).</li><li>Smith, S. B., Cui, Y., 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=Overstretching+B-DNA:+The+Elastic+Response+of+Individual+Double-Stranded+and+Single-Stranded+DNA+Molecules.">Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules.</a> Science. 271 (5250), 795-799 (1996).</li><li>Wang, 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=Shear-Induced+Extensional+Response+Behaviors+of+Tethered+von+Willebrand+Factor.">Shear-Induced Extensional Response Behaviors of Tethered von Willebrand Factor.</a> Biophysical Journal. 116 (11), 29092 (2019).</li><li>Carlsson, C., Johnsson, M., &#197;kerman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+bands+in+DNA+gel+electrophoresis+caused+by+bis-intercalating+dyes.">Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.</a> Nucleic Acids Research. 23 (13), 2413-2420 (1995).</li><li>Collins, B. E., Ye, L. F., Duzdevich, D., 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=DNA+curtains:+Novel+tools+for+imaging+protein&#8211;nucleic+acid+interactions+at+the+single-molecule+level.">DNA curtains: Novel tools for imaging protein&#8211;nucleic acid interactions at the single-molecule level.</a> Methods in Cell Biology. 123, 217-234 (2014).</li><li>Green, E. C., Wind, S., Fazio, T., Gorman, J., Visnapuu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Curtains+for+High-Throughput+Single-Molecule+Optical+Imaging.">DNA Curtains for High-Throughput Single-Molecule Optical Imaging.</a> Methods in Enzymology. 472, 293-315 (2010).</li></ol>

To obtain high quality data of single-molecule conformational changes using fluorescence microscopy as described in this method, it is critical to incubate the molecule for the appropriate amount of time, minimize its nonspecific interactions with the surface and establish microscope settings that reduce photobleaching. The ability of the molecule to freely change conformation is related to the number of biotin-streptavidin interactions formed between the molecule and the surface. As mentioned previously, this must be co...

Mechanisms of how biomolecules respond to environmental stimuli have been studied widely. In a flow environment in particular, shear and elongational forces regulate the conformational changes and potentially the function of biomolecules. Typical examples include shear-induced unraveling of lambda DNA and von Willebrand factor (VWF). Lambda DNA has been used as a tool to understand conformational dynamics of individual, flexible polymer chains and the rheology of polymer solutions1,2,3,4. VWF is a natural flow sensor that aggregates platelets at wound sites of blood vessels with abnormal shear rates and flow patterns. Unraveling of VWF is essential in activating the binding of platelets to the A1 domain and collagen binding to the A3 domain. In addition, high shear-induced A2 domain unfolding allows the cleavage of VWF, which regulates its molecular weight distribution in circulation5,6. Thus, direct visualization of how these molecules behave under flow can greatly enhance our fundamental understanding of their biomechanics and function, which in turn can enable novel diagnostic and therapeutic applications.
Typical methodologies to characterize single-molecule conformations include optical/magnetic tweezers, atomic force microscopy (AFM) and single-molecule F&#246;rster resonance energy transfer (FRET)7. Single-molecule force spectroscopy is a powerful tool to investigate the force and motion associated with the conformational changes of biomolecules. However, it lacks the ability to map overall molecular conformations8. AFM is capable of imaging with high spatial resolution but is limited in temporal resolution9,10. In addition, contact between the tip and the sample may confound the response induced by flow. Other methods like FRET and nanopore analytics determine single-molecule protein folding and unfolding states based on the detection of intramolecular distance and excluded volumes. However, these methods are still in their infancy and limited in their direct observation of single-molecule conformations11,12,13,14.
On the other hand, directly observing macromolecules with high temporal and spatial resolution under fluorescence microscopy has improved our understanding of single-molecule dynamics in many biological processes15,16. For example, Fu et al. recently achieved simultaneous visualization of VWF elongation and platelet receptor binding for the first time. In their work, VWF molecules were immobilized on the surface of a microfluidic channel through biotin-streptavidin interactions and imaged under total internal reflection fluorescence (TIRF) microscopy at varying shear flow environments17. Applying a similar method as Fu&#39;s, we here demonstrate that conformations of VWF and lambda DNA can be directly observed under both TIRF and confocal fluorescence microscopy. As shown in Figure 1, microfluidic devices are used to create and control shear flow, and biomolecules are immobilized on the channel surface. Upon the application of varying shear rates, conformations of the same molecule are recorded to measure the extensional length, also shown in Figure 1. The method could be widely applied to explore other polymer behaviors under complex flow environments for both rheological and biological studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 Labeling Kit</td>
			<td>Invitrogen</td>
			<td>A30006</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Spin P-6 Gel Columns</td>
			<td>Bio-Rad</td>
			<td>7326221</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4501</td>
			<td>Use as free biotin in Step 5.6</td>
		</tr>
		<tr>
			<td>Biotin-14-dCTP</td>
			<td>AAT Bioquest</td>
			<td>17019</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>A8549</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-195</td>
			<td>No. 1 &#189;, 22 x 50 mm</td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr>
			<td>Float Buoys for Mini Dialysis Device</td>
			<td>Thermo Scientific</td>
			<td>69588</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow Fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td>New England BioLabs</td>
			<td>M0212S</td>
			<td>Use for 10X reaction buffer in Step 2.1.1 and 1X reaction buffer in Step 2.2.2</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England BioLabs</td>
			<td>N3011S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Dialysis Device</td>
			<td>Thermo Scientific</td>
			<td>69570</td>
			<td>10K MWCO, 0.1 mL volume</td>
		</tr>
		<tr>
			<td>NEBuffer 4</td>
			<td>New England BioLabs</td>
			<td>B7004S</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-PEG4-Biotin</td>
			<td>Thermo Scientific</td>
			<td>21330</td>
			<td></td>
		</tr>
		<tr>
			<td>Protocatechuate 3,4-Dioxygenase</td>
			<td>Sigma-Aldrich</td>
			<td>P8279</td>
			<td></td>
		</tr>
		<tr>
			<td>Protocatechuic acid</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-205818</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Elastomer Kit for PDMS Fabrication</td>
			<td>The Dow Chemical Company</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sigma-Aldrich</td>
			<td>85878</td>
			<td></td>
		</tr>
		<tr>
			<td>The Blocking Solution</td>
			<td>CANDOR Bioscience</td>
			<td>110 050</td>
			<td>Use as casein blocking solution throughout protocol</td>
		</tr>
		<tr>
			<td>Vinyl Cleanroom Tape</td>
			<td>Fisher Scientific</td>
			<td>19-120-3217</td>
			<td></td>
		</tr>
		<tr>
			<td>von Willebrand Factor, Human Plasma</td>
			<td>Millipore Sigma</td>
			<td>681300</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1 Dye</td>
			<td>AAT Bioquest</td>
			<td>17580</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mm Inner Diameter Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-06419-00</td>
			<td></td>
		</tr>
		<tr>
			<td>25 Gauge Needle</td>
			<td>Thomas Scientific</td>
			<td>JG2505X</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing single-molecule conformational changes under shear flow with fluorescence microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

Observing the dynamic behavior of biomolecules such as VWF and lambda DNA is highly dependent on optimizing their binding to the device surface. Incubating surface treatments for the recommended times in the microfluidic device is crucial to obtaining binding with a few anchorage points, so that molecules can freely extend and relax upon changing flow. If the proteins or DNA are bound too strongly with multiple linkages, they will either extend to limited lengths or not extend at all. This occurs particularly with VWF wh...

Watch this Scientific Journal Video about Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy at JoVE.com

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

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

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

Quantifying the conformational changes in single bio-molecules under shear flow is useful for understanding the function and biophysics of macromolecules like protein and DNA. Fluorescence microscopy allows real-time in-situ visualization of single molecules in physiological flow environments with high temporal and spatial resolution. This is unmatched by other techniques like AFM.By characterizing the shear force under which proteins our DNA have global conformational changes, one can better design drugs that mimic these biophysical properties. This protocol can be widely applied to study the behavior or other polymers, especially bio polymers, in varying flow conditions and to investigate the rheology of complex fluids. The timing of steps is critical to success.We suggest practicing the microfluidic device surface treatment and fluorescence microscopy steps many times to execute them in an efficient fashion. Capturing conformational changes in biomolecules is a visual and dynamic phenomenon best viewed on film. Scientists will better understand this method after seeing the real-time protein and DNA microscopy presented in this video.To prepare the microfluidic device, add five parts of silicone elastomer base to one part curing agent in a weigh boat. and stir the contents thoroughly for one minute to create a pre-cured PDMS solution. Place the master silicon wafer in a plastic Petri dish and pour the PDMS solution over the wafer to create a five-millimeter layer.Cover the dish and leave it in a desiccator under vacuum for one hour to remove air bubbles. Then incubate the covered Petri dish at 60 degrees Celsius overnight to cure the PDMS into a flexible solid, which will result in microfluidic channels being molded into the PDMS at the PDMS wafer interface. On the next day, use a razor to cut 20 by 10 millimeter rectangles around each microfluidic channel in the PDMS and remove the rectangular blocks with tweezers.Use a 25-gauge blunt end needle with sharpened edges to punch a 0.5 millimeter diameter hole at one end of the channel, making sure that the hole goes completely through the PDMS block. Use a thin needle to punch the PDMS out of the hole and repeat the process at the other end of the channel. Place the PDMS block with the channel side up and the coverslip into the chamber of a plasma bonding machine and start the treatment.When treatment is complete, quickly place the PDMS block on a coverslip so that the channel is in contact with the slip. Apply pressure to the edges of the block, then place the coverslip PDMS assembly on a hot plate at 115 degrees Celsius for 15 minutes to reinforce the permanent bond. Finally, insert a 10-centimeter long.0.25 millimeter inner diameter tube into the outlet hole at the top of the PDMS block to allow fluid to easily flow out of the channel. For von Willebrand factor or VWF experiments inject up to 10 microliters of 10 micrograms per milliliter BSA-Biotin dissolved in sterile 1X PBS into the inlet of the microfluidic device. Withhold a few microliters of BSA-Biotin in the pipette tip after injection and allow the tip to remain embedded in the inlet.Allow the BSA-Biotin to incubate in the device for two hours which will result in BSA non-specifically binding to the coverslip surface. Then remove the pipette tip and inject up to 10 microliters of casein blocking solution into the channel. Allow the casein solution to incubate for 30 minutes so it can block any free sites and reduce nonspecific binding of the biomolecules to the surface.Remove the tip and inject up to 10 microliters of streptavidin in sterile PBS into the channel, which will bind to the biotin groups of the BSA-Biotin. Next, inject up to 10 microliters of detergent solution to wash away excess streptavidin. Remove the tip and inject either VWF in casein solution or lambda DNA.Incubate VWF for three minutes or lambda DNA for 45 minutes. Then inject five millimolar free biotin to block the excess streptavidin binding sites. Load one milliliter of casein blocking solution into a syringe and secure it in a syringe pump.Then attach one end of the tubing to the syringe needle and flow in the solution to remove air bubbles. Approximately three minutes after the free biotin injection attach the other end of the tube to the inlet of the microfluidic device. Select the highest magnification objective of the TIRF or confocal fluorescence microscope.If needed, add a drop of immersion oil on the objective. Place the microfluidic device on the microscope stage so that the coverslip is flush with the objective. Start bright-field microscopy and adjust the focus so that features such as debris and bubbles are visible.Then adjust the stage in the X and Y directions until the edge of the microfluidic channel is visible and bisects the frame. Switch to the FITC channel and adjust the Z level and TIRF angle as needed until the individual green globular molecules can be distinguished. Adjust laser intensity and exposure time to visualize fluorescent molecules without photobleaching them too quickly.Then adjust the contrast to better visualize the molecules. Exactly five minutes after free biotin injection start and stop flow from the syringe pump to observe the changes in molecular conformation using flow rates between 5000 and 30, 000 microliters per hour. With VWF it is critical to apply at least a small amount of flow, such as 30 seconds of 10, 000 microliter per hour flow exactly five minutes after free biotin injection.Repeat this throughout various areas of the microfluidic device and locate molecules that can extend and relax upon multiple cycles of starting and stopping flow. Note how long it takes for molecules to reach maximum extension and complete relaxation and record videos of the continuous behavior of molecules under shear flow. The flexibility of a molecule to change confirmation is often demonstrated by its ability to lengthen as higher shear rates are applied due to increasing flow.The extension versus shear rate curve of a von Willebrand factor molecule is useful for characterizing the biomechanical properties of the protein. Fluorescence microscopy images of lambda DNA similarly show increased extension upon higher shear rates at 30 seconds and gradual relaxation over two minutes after flow is stopped. When working with VWF, remember to incubate it only for three minutes and free biotin for five minutes before starting flow.This is critical for forming the optimal number or biotin-streptavidin linkages needed for reversible unraveling. This method can be adapted to visualize real time interactions between multiple biomolecules and characterize their functions. For example, one could better understand platelet plug formation by visualizing VWF unraveling under high shear and its binding with the platelet adhesion molecule GPIb-alpha with this method.

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Bioengineering

5.7K visualizações.  Lehigh University. Quantificar as alterações conformais em biomoléculas únicas sob fluxo de cisalhamento é útil para entender a função e a biofísica de macromoléculas como proteína e DNA. A microscopia de fluorescência permite a visualização em tempo real in situ de moléculas únicas em ambientes de fluxo fisiológico com alta resolução temporal e espacial. Isso é incomparável por outras técnicas como a AFM.Ao caracterizar a força de tesoura sob a qual as proteínas do nosso DNA têm...