{"id":392,"date":"2016-08-28T03:09:16","date_gmt":"2016-08-28T03:09:16","guid":{"rendered":"http:\/\/biodigestor.net\/?p=392"},"modified":"2016-08-28T03:09:16","modified_gmt":"2016-08-28T03:09:16","slug":"we-report-the-development-of-a-simple-to-implement-magnetic-force-transducer-that","status":"publish","type":"post","link":"https:\/\/biodigestor.net\/?p=392","title":{"rendered":"We report the development of a simple-to-implement magnetic force transducer that"},"content":{"rendered":"

We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA\u2013protein complexes in the horizontal plane. magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be Flumazenil used to study DNA binding proteins at the single molecule level. Flumazenil is the applied force is Boltzmann\u2019s constant is the absolute temperature of ~297\u00b0K is the persistence length of 50 nm is DNA\u2019s observed end-to-end extension. Our results recapitulate DNA\u2019s mechanical response in the Flumazenil 0.1\u201310 pN range (1). These data were obtained over 45 min; however experiments can last several hours. We find that the bead aspiration buffer volume and other experimental conditions can be stably maintained for this duration. The inset to Figure 2B shows the transition from DNA\u2019s entropy-dominated response to the Hookean elastic response; the stars and squares are from experiments performed at 0.320 \u03bcm\/s while the diamonds are data from an experiment performed at 1.6 \u03bcm\/s. Overall we see excellent agreement between the force-extension data and the worm-like-chain model across the range of forces for which the model is valid. The steps involved in these experiments are described in Section 9 of the Supplementary Materials. Micromechanical experiments designed to study protein dissociation as a function of force require a method for adjusting the tension on protein-loaded DNA tethers. For this the force must be changed slowly enough to leave the protein\u2013DNA complex in equilibrium. A prerequisite for this is to be able to adjust tension on protein-free DNA while ensuring equilibrium. To test for the reversibility of force loads we repeatedly extended and contracted a DNA molecule with no bound proteins. As Figure 3A shows there is minimal hysteresis indicating that forces can be adjusted while leaving DNA tethers in equilibrium. From these data we extrapolate to the case of tethers with bound proteins. An extrapolation is necessary since we used histones which unbind irreversibly beyond a certain force implying that maintenance of binding\u2013unbinding equilibrium as a function of force loading rates could not be tested directly. However when we performed experiments with histones we found that the measured critical force and other quantities agreed Flumazenil well with bulk experiments (where available) or theoretical estimates premised on the presence of equilibrium. Thus we conclude that the loading rates achievable in our instrument minimally disturb bound proteins. Figure 3 Testing for hysteresis force calibration and precision Force measurements obtained from our fluctuation-dissipation method were verified as follows. Using micropipettes with a 15\u201320 \u03bcm opening we released Rabbit polyclonal to AADACL3.<\/a> 2.8 \u03bcm magnetic beads 300 \u03bcm from the magnet and halfway between the floor and the roof of the cell and then at distances from the magnet increasing in 100 \u03bcm increments up to 2500 \u03bcm. The buffers used were a low viscosity 1.5 centi-Poise (cP) 25 w\/v CaCl2 solution and a high viscosity 7 cP 55% w\/v glycerine (glycerol) solution (11). Magnetic particles (bead density ~1.22 g\/cm3) are neutrally buoyant in the CaCl2 solution while glycerine retards sedimentation. The beads quickly reached terminal velocity. The spatial rate of change of the component of the magnetic field pointing toward the magnet does not vary too greatly over a distance of 20\u201330 \u03bcm as estimated by the constancy of force over 20\u201330 \u03bcm changes in distance between tethered beads and the magnet Flumazenil<\/a> as close as Flumazenil 300 \u03bcm from the magnet which is also the approximate is the velocity in the direction of the force and is the bead diameter (2.8 \u03bcm). Because the velocity of the beads is ~10 \u03bcm\/s and thus the Reynolds number is small use of Stokes\u2019 drag law is valid. Furthermore the effect of the vertical bounding surfaces is negligible because particle trajectories are confined to a plane well separated from them. The velocities were calculated using a custom particle tracking software described in the Supplementary Material. The buffer viscosities were measured using a Thermo Haake RheoStress 600 viscometer (Thermo Scientific Pittsburgh PA). Figure 3B shows the results of these calibration experiments. The three dashed curves and one thick solid curve each represent a calibration experiment while the thin solid curve represents the average.<\/p>\n","protected":false},"excerpt":{"rendered":"

We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA\u2013protein complexes in the horizontal plane. magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can… Continue reading We report the development of a simple-to-implement magnetic force transducer that<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[77],"tags":[389,388],"class_list":["post-392","post","type-post","status-publish","format-standard","hentry","category-7-tm-receptors","tag-flumazenil","tag-rabbit-polyclonal-to-aadacl3","entry"],"_links":{"self":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/392"}],"collection":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=392"}],"version-history":[{"count":1,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/392\/revisions"}],"predecessor-version":[{"id":393,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/392\/revisions\/393"}],"wp:attachment":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=392"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=392"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=392"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}