Name: Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins (Video) | JoVE Journal
Uploaded: 2011-03-19T11:00:00.000+00:00
Duration: 13 min 51 s
Description: Learn to operate a gravitational force spectrometer for single-molecule force measurements using light microscopy and digital imaging. This method enables precise molecular interaction analysis in neuroscience and biophysics research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This material is based upon work supported by the National Science Foundation under Grant No. 0842736.

Introduction to the Novel GFS Configuration
 The GFS consists of several essential components: A regular light microscope, an equatorial mount, a camera, and a computer [Figure 1]. The sealed flow-cell chamber which holds the sample is also indispensible according to the GFS design. The Light microscope is mounted onto the equatorial mount so the scope can be rotated into different orientations in space. This ability allows the static vector of gravity to be exploited so that the samples can be dynamically oriented in relation to the vector so the force of gravity can impart piconewton-range force loads to the samples. The camera replaces the light microscope's ocular lens so it can record changes in the orientation of the sample. This raw data is digitized and manipulated by the computer to interpret the data into actual force and distance measurements. The sealed flow-chamber is designed to allow all degrees of freedom in space without sample loss. In the chamber resides the sample molecule which is tethered near one terminus to an "anchored" bead that is glued to the surface of the chamber. The opposite terminus is tethered to a "mobile" bead that is free from the surface of the chamber. It is this mobile bead, free in the assay buffer that can be acted upon by gravitational force thereby stretching the tethered molecule at such low force loads [Figure 2]. The sample simply looks like a pair of microspheres under the microscope, although it does take some experience to discern good usable pairs coupled by their attachment to a molecule from pairs where both beads are sitting on the surface of the flow-chamber. One modification to the system is the addition of a floating platform which holds the GFS and is suspended by springs. In this configuration, once the sample has been rotated into a position where gravitational force can act on the sample, the platform and all its components can be dropped against the spring constant. Near free-fall, the force acting on the mobile bead is close to zero and at the springs' maximum extension, the gravitational force is multiplied by as much as two times. In this way, a graded force/distance response can be graphed to measure the behavior of a single molecule at different force loads.
1. Microsphere Preparation
<ol>
<li>Submerge about 10 mg of glass or silica beads in 0.04% 3-Aminopropyltriethoxysilane (cut with acetone) for two minutes.</li>
<li>Rinse with two changes of double distilled water and centrifuge to pellet at 2000 X G for 5 minutes. Discard supernatant.</li>
<li>Add 5 mL of coupling buffer (0.01 M pyridine cut with double distilled water and adjust pH to 6.0), and shake this mixture vigorously. Centrifuge as described above. Repeat this step three times.</li>
<li>To wet cake of beads, add 2 mL of 5% gluteraldehyde solution (cut gluteraldehyde with coupling buffer). Shake vigorously.</li>
<li>Under a hood, rotate bead/gluteraldehyde mixture for 3 hours at room temperature.</li>
<li>Centrifuge as above and aspirate the supernatant.</li>
<li>Wash the beads in 5 mL of coupling buffer by shaking them vigorously and centrifuge and aspirate the supernatant. Repeat this three more times.</li>
<li>Add about 15 μL of the desired antibody to beads and shake vigorously. The beads should be rotated for 16-24 hours.</li>
<li>Under the hood, add 5 mL of 1 M glycine quenching solution (cut glycine with double distilled water and adjust pH to 7.0). Shake this mixture vigorously and rotate for 30 minutes.</li>
<li>Centrifuge and aspirate the supernatant.</li>
<li>Add 5 mL of wash buffer (0.01 M Tris, pH 7.0; 0.1% sodium azide; 0.1% BSA; 0.15 M NaCl; and 0.001 M EDTA). Shake this vigorously, centrifuge and aspirate the supernatant. Repeat this step three additional times.</li>
<li>Change buffer to low-salt buffer (0.1 M KCl; 0.02 M imidazole; 5 mM MgCl2; adjust to pH 7.0). Repeat three times.</li>
</ol>
2. Sample Attachment to Microspheres
<ol>
<li>Take a small amount of prepared beads (about 2 μL from each cake of beads however, if there is a large discrepancy in diameter between batches, it is advantageous to use around an 8:1 ratio of large to small beads) and add them to a microcentrifuge tube with assay buffer. Reduce the concentration of your protein to around 5 μM, using the assay buffer. Prepare at least a total volume of 400 μL including the buffer, the protein, and the beads.</li>
<li>Rotate this mixture at around 1 RPM for 3 hours (if the protein is agitated too much, it will aggregate and become useless).</li>
</ol>
3. Slide Chamber Preparation
<ol>
<li>Coat a thick microscope slide with 0.01% nitrocellulose (in amyl acetate). Let this slide dry for about 10 minutes.</li>
<li>With a sharp glass cutter, cut cover slide so as to make a chamber. This requires four strips of glass.</li>
<li>Use the factory edge of the glass and rake it across a smear of vacuum grease on both sides of the edge.</li>
<li>Press grease coated strips onto dried nitrocellulose covered slide to create a box on the surface of the slide.</li>
<li>Pipette about 2 μL of bead/protein mixture by tapping pipetter onto surface of glass inside the box.</li>
<li>Add about 20-400 μL of low salt buffer depending on how large the chamber is.</li>
<li>Press a cover slip on top of the box which is already coated with vacuum grease to finish the sealed and buffered chamber.</li>
<li>Let slide sit on a level place so the beads have ample time to anchor themselves to the nitrocellulose.</li>
</ol>
4. GFS Data Acquisition
<ol>
<li>Mount slide onto GFS stage</li>
<li>Monitor what the GFS camera is recording and search for legitimate "bead pairs," in which the small microsphere is attached near the equator of the large microsphere.</li>
<li>When a potential bead pair is found, go through the depth of focus to determine if the "mobile" bead is not resting on the surface of the slide.</li>
<li>Once a suitable pair is identified, note angle mobile bead is away from dmax (dmax = the maximum distance between the centroids of the coupled microspheres). Move the scope into position to acquire video.</li>
<li>The angle of travel of the GFS should be sufficient to record dmin, dmax, and dmin which might call for 25-90 degrees depending on the length of the molecule.</li>
<li>RECORD as the pair travels from dmin to dmax and back to dmin.</li>
<li>It is a good idea to also shoot a movie of the background so it can be subtracted later for analysis.</li>
<li>If performing a GFS drop, move the scope back to dmax and record the drop at at least 60 frames per second. The critical part is the first oscillation, but long record times can also be used for dynamic study.</li>
</ol>
5. GFS Data Analysis
<ol>
<li>Transform raw video into digitally "thresholded" image and run macro in imageJ to determine the centroid position of each bead in every frame of video. This is also for the drop video.</li>
<li>Dump the X,Y and area data from ImageJ into Excel and plot the points.</li>
<li>If a proper bead pair was acquired by video, a noticeable hump in the graph is indicative of the beads being at their closest (dmin) and at the apex of the graph is the position of dmax.</li>
<li>Using this data, the radius of each bead should be determined precisely in ImageJ and all this information is put into the nested equation: 
d = [(g sin α)2 + (g cos α + dmax - g)2]0.5 
g = [dmin2 + rb 2 - (ra + rb)2]0.5 = rb sin β 
c = dmax - ra - g 
(d = distance between centroids; g = force of gravity; α = the angle in degrees parallel to the objective lens; rb = radius of mobile bead; ra = radius of anchored bead; β = angle of attachment off the axis of the equator of the anchored bead.</li>
<li>Using the fitted radius of the mobile bead which also yields its volume, and given the density of the bead, the force the mobile bead imparts on the molecule can be calculated in piconewtons after the buoyancy of the solvent is subtracted. This method measures the force on the tethered molecule in piconewtons and calculates the absolute molecule length between antibody attachments in nanometers. F = V (d-b) a (F = force, V = volume, d = density of the glass microsphere, a = acceleration due to gravity, b = the density of the displaced water). </li>
</ol>
6. Representative Results:
If the bead prep is done correctly, there will be minimum bead aggregation although there may still be an occasional bead clump. When viewed through the scope, there should be a reasonable distribution of beads whether paired or not in the chamber.
It is important to minimize vibrations as much as possible; to do this either an air table, special shock absorbing feet for a tripod that holds an EQ mount, or on the system that utilizes springs can be used for vibration isolation.
Another useful tip concerning the sealed flow chamber is to let it stand for about five minutes on a level table after it has been fully constructed. This allows any unattached larger beads to float down through the buffer and rest in the layer of nitrocellulose. If the slide were instead mounted directly upon completion, the investigator would continually have to deal with beads literally flying through the field of view; and if this happens during video acquisition it can corrupt the experiment. If this is done properly, bead flight is significantly minimized and cleaner video results.
When a potential bead pair is identified by the GFS operator, it is useful to put it through a preliminary rotation to monitor the behavior of the pair. Rarely, the large bead is not firmly affixed to the slide. If this happens, there is no use in utilizing the pair because it is critical that the larger anchored bead stay in a fixed position through the duration of the acquisition. If the pair is stable and does not exhibit "anchored bead roll," then it is suitable for experimentation.
A plot of bead separation distances versus change in angle relative to gravity can be used to evaluate the acquired data. A good representative result would show little separation on a plateau and as the mobile bead frees itself from the anchored bead because of the influence of gravity, the graph will show more separation. This continues until a nice peak is reached which is called dmax and the curve starts downward again returning to the baseline dmin [Figure 1]. In ideal conditions, this signature is symmetrical. A non-representative result would show a graph with incoherent patterns showing no distinct dmin-dmax-dmin pattern; or it would show separations that are orders of magnitude too high for a single molecule indicating maybe there was a piece of dust between the beads, or maybe that the mobile bead was simply not attached at all. The process of finding the pair, shooting it, processing it, and analyzing it has many stop gaps where improper bead pairs are culled out. So, if you get to the end of the entire process and you have a molecule length that is consistent with previous results, one can be very confident that the original bead pair is representative and can be included in the results. On a good day, about half of the bead pairs shot can be taken through to become a legitimate data point. For instance, the length of the coiled coil of myosin between MF20 and MF30 antibodies, is known based on EM data and AFM, data to approach 100 nm 2,7,8,9. If the result is several times this length, the sample has aggregated. The results presented here are from standard GFS rotation experiments and demonstrate a distance of 96 nm ± 5 nm, [Figure 2] which agrees closely with the measurements of the MF30 (which binds at the N-terminus of myosin subfragment-2) and MF20 (which binds in the light meromyosin) antibody separation distance on myosin deduced from the literature values [Figure 3].
<img src="/files/ftp_upload/2624/2624fig1.jpg" alt="Graph of bead centroid distances vs angle, showing mobility analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. GFS configuration. Major parts of GFS are labeled.
<img src="/files/ftp_upload/2624/2624fig2.jpg" alt="Rotational dynamic analysis, diagram with GFS rotation, gravity vector, experimental setup for study." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Schematic of GFS principle. Left side shows centroid of mobile bead at a minimum distance from centroid of anchored bead. As GFS is rotated, mobile bead aligns with vector of gravity which is also running parallel with the axis of the tethered molecule. In this position, the distance between the centroids of the mobile and anchored beads is at a maximum. Top right shows a representative slice from a GFS movie. Bottom right is an image of the GFS undergoing rotation.
<img src="/files/ftp_upload/2624/2624fig3.jpg" alt="Static equilibrium graph; distance vs. angle; data analysis; measurements in microns, radians." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Representative result showing dmin on the left of the graph; dmax at a relative separation of 17.78 microns; and a return to dmin around the baseline of about 17.75 microns of relative separation between the beads.
<img src="/files/ftp_upload/2624/2624fig4.jpg" alt="Static equilibrium graph showing force vs. cord length in nanometers, data analysis." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Representative result of GFS rotation experiment showing distance between MF20 and MF30 antibodies average 96 nm. This represents the approximate length of S2.
<img src="/files/ftp_upload/2624/2624fig5.jpg" alt="Myosin-actin interaction diagram, showing antibodies MF20, MF30, protein structure, hinge points." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. Myosin II dimer used to show possible attachments for use with the GFS including antibody and/or actin attachments. Different attachment possibilities allows for different GFS strategies to measure different regions, or to apply force perpendicular or parallel to the rod domain of the myosin dimer.

<ol><li>Schwaiger, I., Sattler, C., Hostetter, D. R., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myosin+coiled-coil+is+a+truly+elastic+protein+structure%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">The myosin coiled-coil is a truly elastic protein structure.</a> Nat. Mater. 1, 232-235 (2002).</li>
<li>Root, D. D., Yadavalli, V. M., Forbes, J. G., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coiled-coil+nanomechanics+and+uncoiling+and+unfolding+of+the+superhelix+and+alpha-helices+of+myosin%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin.</a> Biophysical Journal. 90, 2852-2866 (2006).</li>
<li>Nishizaka, T., Miyata, H., Yoshikawa, H., Ishiwata, S., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unbinding+force+of+a+single+motor+molecule+of+muscle+measured+using+optical+tweezers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Unbinding force of a single motor molecule of muscle measured using optical tweezers.</a> Nature. 377, 251-254 (1995).</li>
<li>Gawalapu, R. K., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+labeling+and+computational+analysis+of+the+strut+of+myosin%27s+50+kDa+cleft%5BTitle%5D)+AND+%22Arch.+Biochem.+Biophys%22%5BJournal%5D)">Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft.</a> Arch. Biochem. Biophys. 456, 102-111 (2006).</li>
<li>Kellermayer, M. S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+and+manipulating+individual+protein%5BTitle%5D)+AND+%22Molecules+Physiol.+Meas%22%5BJournal%5D)">Visualizing and manipulating individual protein.</a> Molecules Physiol. Meas. 26, R119-R153 (2005).</li>
<li>Shimizu, T., Dennis, J. E., Masaki, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axial+arrangement+of+the+myosin+rod+in+vertebrate+thick+filaments%3A+immunoelectron+microscopy+with+a+monoclonal+antibody+to+light+meromyosin%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.</a> J. Cell Biol. 101, 1115-1123 (1985).</li>
<li>Godfrey, J. E., Harrington, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-association+in+the+myosin+system+at+high+ionic+strength.+I.+Sensitivity+of+the+interaction+to+pH+and+ionic+environment%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment.</a> Biochemistry. 9, 886-893 (1970).</li>
<li>Root, D. D., Stewart, S., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+docking+of+myosin+and+actin+observed+with+resonance+energy+transfer%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Dynamic docking of myosin and actin observed with resonance energy transfer.</a> Biochemistry. 41, 1786-1794 (2002).</li>
<li>Xu, J., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conformational+Selection+during+Weak+Binding+at+the+Actin+and+Myosin+Interface%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Conformational Selection during Weak Binding at the Actin and Myosin Interface.</a> Biophys. J. 79, 1498-1510 (2000).</li>
<li>Sattin, B. D., Pelling, A. E., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+base+pair+resolution+by+single+molecule+force+spectroscopy%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">DNA base pair resolution by single molecule force spectroscopy.</a> Nucleic Acids Res. 32, 4876-4883 (2004).</li>
</ol>

No conflicts of interest declared.

When converting a movie to a digitally thresholded representation, it is crucial for the thresholded image to maintain the same area in each frame of video. Because the beads in a bead pair move independently of one another, any drift in the thresholded areas can also cause the relative distances between the centroids of the beads to drift and introduce significant error. Controlling the threshold area reduced the error five-fold in the distance measurements from 26 nm down to 5 nm. It is also crucial to obtain an acc...

<table><tbody><tr><td>3-Aminopropyltriethoxysilane</td><td>Polysciences, Inc.</td><td>919-30-2</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A18P-4</td><td></td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>110-86-1</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Fisher Scientific</td><td>G7776</td><td></td></tr><tr><td>Glycine</td><td>Research Organics</td><td>BP381-1</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>9682T</td><td></td></tr><tr><td>Sodium azide</td><td>Amresco</td><td>71289</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>AMR-0332-100G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>EDTA</td><td>MSI</td><td>E9884</td><td></td></tr><tr><td>Nitrocellulose</td><td>Sigma-Aldrich</td><td>60443</td><td></td></tr><tr><td>N-N Dimethyl Formamide</td><td>Extracted from Large New</td><td>D4254</td><td></td></tr><tr><td>Rabbit skeletal myosin II</td><td>Zealand White Rabbits (7-8)</td><td>NA</td><td></td></tr><tr><td>MF30 antibody (9-10)</td><td>Developmental Studies Hybridoma Bank</td><td>MF30</td><td></td></tr><tr><td>MF20 antibody (6)</td><td>Hybridoma Bank</td><td>MF20</td><td></td></tr><tr><td>Lab microscope</td><td>Boreal</td><td>WW57905M00</td><td></td></tr><tr><td>Equatorial mount</td><td>Celestron</td><td>CG-5</td><td></td></tr><tr><td>Digital video cam</td><td>Sony Corporation</td><td>XCDV60</td><td></td></tr><tr><td>Caliper release</td><td>Cabelas</td><td>IA-415482</td><td></td></tr><tr><td>Compression spring</td><td>Jones Spring Co.</td><td>723</td><td></td></tr><tr><td>Extension spring</td><td>Jones Spring Co.</td><td>770</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>NA</td><td></td></tr><tr><td>Fire-i drivers &amp;amp; application </td><td>Unibrain</td><td>3.80</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td>NA</td><td></td></tr></tbody></table>

demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

The gravitational force spec consists of an epi mount, a regular light microscope, digital camera, and a computer. We use this to do single molecule acid. We can record force equilibrium or we can provide a grade force by drop.To start, we need to prepare a glass micro so they can done by these type of process. To measure out about 10 milligrams of the beans that you gonna use, I usually prepare 2K. Here we have five milliliters of we're gonna three triology.That's where we're gonna get our terminal Put One. Go ahead and mix that up really nice with a transfer and then transfer two milliliters of the mixture on top of your be cake. Leave the beep cakes for two and then vortex thoroughly at two.The down ate the wet two. You want oh one purine in double distilled water. pH six coupling.So you simply add five milliliters of the coupling buffer to each beef and this coupling make the bead to ling. So then antibodies the closeup floating ling again, vortex for a minute. Pin them down for five minutes and if the freezer to get your glide, you're gonna cut it down from whatever extent it's to 5%using a coupling buffer.Then you take two milliliters of this solution add and this will allow the TER couple with the terminal that are already on our vortex. This for two, rotate for three. You coupling buffer procedure.Again five milliliters, a coupling buffer into your feed cake. Again, mix, spin and aspirate.Sate. On the fourth time some sate on the top of the cake, the antibody to freely move around.This is our antibody here. Micro that volume. The wet rotate antibodies, you'll seven which is one moly cut with double distilled water and the mixture is rotated under the for 30 minutes for the appropriate time.To quench all the underact glue, eye to eye. Gonna spin it down, get rid of the S.Then you wanna make a the contents of which are protocol. Take five milliliters of the take the beads, here's the beads, intex down tooth.Repeat the step three times and then to whatever assay you're doing the same. To start our preparation, we first need to get our protein. In this case go to the negative 70, the keep stored protein, it's important to this protein and heat it up.This now this right outta the, is gonna be much more concentrated what we wanna use in our assay. So the cut down to about fifteens and gonna put that out some into the tip. But then that has to be because which accurate volume after you out the volume amount down to about five micro this in the large beads, we're gonna take bigger volume, gonna be 10 microliters from this example bead.And that's gonna give a nice, here we're adding about three 50 more microliters assay buffer into the microcenter tube to give us a final concentration. So now we're going to the do it vi like with aex your will A here we're rotate the one literally and we'll do that. Now it's time to actually construct the flow on which our fees will reside and be.So I'm putting this standard microscope slide into oh 1%and that be cut with a glue for the anchorings to attach to the surface of the, the glass cut and cover and microscope slides. I've already edges with the, and I'll apply this thinner piece of glass thicker hyper slide. This chamber will be relatively small.So here's a small here And it's damaged position's. Another one of those Guys hold Every day. The agent makes me better, lack better.Here we'll pass down the last two 25-year-old possibly taste before we start adding cracker and the cheese. And you've missed sun. Only a few have taste in just a few.And here if you look closely you can actually see the bead. The tip, what I do is I start by simply tapping on the area I be and I don't, after I do that full while the rest of be the surface of the slide here, we'll at about 40 into be so that we can put this slide through different orientations and not worry about a purposes can migrate down. Hey you don't need no co screw to sample my bouquet for some of my wine, a back of the line and I might get holy Mount the slide and I use an extra clip to keep things more stable And we can put the objective.This is a remote control we use to position of the here we're slide and everything's turned on including the camera. We can start monitoring for potential be do this by simply moving monitoring the video screen. The one on the left has a smaller bead and its smaller bead kind of looks like the other smaller beads that are just sitting there on the surface of the lop.However, the bead pair on the, we has a bead that comes in focus when the off bead surface of the LOP that indicates that that be resides in a different of focus than the other be slide and also pair so that be on the right A shows how pairs might work on a slide. So we put the slide as we go through rotation with different angles that gives us different lengths of, we then use rotation. We see a in of what a feed para might look like using a computer.We find the both. This case calculate we can drop the provide measure and here drop the F and also dropped. Thanks for watching.Good luck with your disturbance. There's an time to dance. Besides, don't think any won't win mine.My friends up busy trying to score it. It's all rod me behind. Where's that girl I came with?She goes leaving with my friend. She really thinks she is. Don't she know real man.Ride again, right again, mechanical bull. Let it let go. Right, right, right down in Mexico.I love the way on fucking rounds tonight. Mechanical bull. Don't you slow down.Give me 5 worth to set me down when you finish with my left hand. Just to use my right. Yeah, with my left hand I'm just gonna use right.

demonstrating-uses-novel-gravitational-force-spectrometer-to-stretch

Research

JoVE Journal

Bioengineering

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr period.

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

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

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Here, we present a protocol for the simultaneous use of Förster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Ensemble force spectroscopy (EFS) is a robust technique for mechanical unfolding and real-time sensing of an ensemble set of biomolecular structures in biophysical and biosensing fields.

Ensemble Force Spectroscopy by Shear Forces

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

Learn to operate a gravitational force spectrometer for single-molecule force measurements using light microscopy and digital imaging. This method enables precise molecular interaction analysis in neuroscience and biophysics research.

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

Explore More Videos