这项工作是支持的巴勒斯惠康在科学界面（ARD），国立卫生研究院通过国立卫生研究院主任的新的创新奖励计划1-DP2-OD007078（ARD）的威廉·鲍斯小斯坦福大学研究生奖学金（ASA的事业奖），斯坦福大学心血管病研究所年轻的博士前奖学金（JC），。作者感谢出借显微镜设备詹姆斯Spudich。

1。流动电池的制备<ol><li>盖玻片（＃1.5，22x22毫米和22x40毫米，厂商VWR）使用超声波清洗。 <ol><li>添加盖玻片盖玻片和装修在sonicator（见步骤2）的一个小玻璃容器。 </li><li>填补与异丙醇的容器和超声在20分钟的沐浴sonicator。 </li><li>丢弃异丙醇和MilliQ Barnsted设备或类似设备由去离子水冲洗量，冲洗盖玻片。装满水的容器中，超声20分钟。 </li><li>超声波处理后，干燥过滤，无粉尘的空气流中的盖玻片。使用只出现干净（即无污迹）的盖玻片。 </li><li>轻轻火焰几秒钟的盖玻片清除任何剩余的灰尘和湿气：通过用本生灯产生的气体火焰盖玻片，照顾，不变形的盖玻片，由于多余的热量。这一步消除表面残留污染物。 </li></ol></li><li>使用双面胶带，连接在一起的两个盖玻片。切〜3厘米长，0.3厘米宽的磁带。留在中间1.6厘米的通道上22x40毫米盖玻片附加的磁带。用枪头轻轻按下盖玻片上，以确保磁带已坚持正确的。 </li></ol> 2。磁性镊子安装和校准<ol><li>磁性镊子安装使用永久稀土永磁材料，描述以前10,12。构建了一个直径1毫米的针孔和一个铝制的L型支架安装在垂直的Z-翻译（Thorlabs）调节高度镊子。两个常任理事国的稀土永磁材料被装针孔创建磁阱（ 图1）两侧的支架。 </li><li>标记λ噬菌体DNA的制备：在其5&#39;和3&#39;的DNA与生物素和地高辛（内幕交易审裁处的DNA，圣迭戈，CA）的两端，用于校准磁遗传镊子。 <ol><li>混合4纳米的λ-DNA 50μL，2μL40生物素标记的寡核苷酸和热量在60°C 10分钟纳米。室温超过1小时，然后慢慢冷静。 </li><li>根据制造商的规格，并加入DNA连接酶在室温下结扎3小时。 </li><li>加入2μL400牛米的地高辛标记的寡核苷酸在室温45分钟孵育。 </li><li>加入1.5μL10 mM的ATP的结扎地高辛标记的寡核苷酸的λ-DNA，并持续3个小时。 </li><li>删除多余的核苷酸和DNA连接酶使用100 kDa的自旋过滤器（单片机Ultracell YM-100）。共有1000倍TE缓冲缓冲区交换是足够的，去除多余的寡核苷酸。注：重要的是不使用自旋速度&gt; 500xG，因为它会导致DNA的剪切。 </li><li>审查通过0.7％琼​​脂糖凝胶电泳对DNA样品，以确保我的DNA不是变性或剪切。 </li><li>测量DNA的浓度。使用一个的NanoDrop紫外 - 可见光谱仪（Thermo Scientific的）建议，因为现有的样本量可能是非常小的。 </li></ol></li><li>第1条所述的准备流细胞。 </li><li>附件流细胞的DNA，加入抗地高辛抗体（羊IgG的1微克毫升-1;罗氏生物科学，曼海姆，德国），并允许它坚持了15分钟的玻璃表面。 </li><li>流细胞表面钝化，以防止非特异性的DNA症结1X PBS中添加2毫克毫升-1牛血清白蛋白，0.1％Tween-20的。在室温下孵育45分钟。 </li><li>重复步骤2.5。 </li><li>新增50时的fuctionalized的λ-DNA，并允许附加15分钟的盖玻片。 </li><li> 1X PBS洗去多余的DNA。 </li><li>加入链霉亲和涂层的超顺磁性珠（1微米微珠= 2微克毫升-1; 2.8微米微珠= 20微克毫升-1 </s达&gt;），并允许他们固定的DNA附上15分钟。 </li><li> 1X PBS洗去多余的珠子。 </li><li>磁镊子校准<ol><li>移动永久磁铁的位置是远离样品表面（&gt; 15毫米）。 </li><li>在显微镜下将准备流细胞。 </li><li>以上的流动池的位置移动到永久磁铁。 </li><li>远离两个玻璃表面上珠为重点，选择焦平面的λ-DNA官能珠。正确的焦平面是在其中所需的珠子有一个光明的中心。 </li><li>采取珠议案的80 Hz或500帧的视频。 </li><li>将磁铁靠近样品表面。对于超过5毫米的距离，移动1毫米递增。 1和5毫米之间的距离，将在0.5毫米递增。对于距离小于1毫米，0.25毫米的增量移动。 </li><li>在每一个新的磁铁的位置，重复步骤2.11.5和2.11.6。 </li><li>对于每个数据设置，使用二维高斯拟合珠跟踪中心。 </li><li>计算通过使用布朗波动的力量： </li></ol><img alt="式（1）" src="/files/ftp_upload/3520/3520eq1.jpg" /> 其中K B T是玻尔兹曼热能，L是λ-DNA的长度，&lt;x 2&gt;质心位置的变化。 <ol start="10"><li>确认通过洛伦兹拟合功率谱检查，以便找到滚降频率的内力计算精度的力量。作用力与滚降频率的关系： </li></ol><img alt="公式（2）" src="/files/ftp_upload/3520/3520eq2.jpg" /> 式中ƒ0是滚降频率， 一个是珠半径，μ为流体粘度12。 </li></ol> 3。胶原蛋白肽扣押流动细胞为了提供一个具体的例子，我们的方法的应用，我们描述了最近在我们的实验室工作，水解胶原蛋白三聚体模型肽的特点。我们预计，这一般方法可广泛应用于其他蛋白质和核苷酸。 <ol><li>胶原蛋白的模型肽由一个 N-端为净化myc基因标记的5倍，（GPP）的10强制三螺旋形成，胶原，6X His-tag的α1残基772-786（GPQGIAGQRGVVGL），从而形成了MMP- 1识别位点中，foldon序列（GSGYIPEAPRDGQAYVRKDGEWVLLSTFL），和一个 C-末端KKCK方便与马来酰亚胺生物素标记。 foldon源于在T4噬菌体蛋白fibritin的，和稳定胶原蛋白模型的三聚体融合的 N -或C-末端13,14。 </li><li>胶原蛋白肽和MMP-1蛋白表达和纯化所述先前4,15。&lt;/ LI&gt; <li>加入抗-myc基因（毫升15微克-1），室温孵育20分钟的流动细胞和允许-myc基因反流细胞表面的重视。 </li><li>钝化流动池，以防止非特异性蛋白质在以同样的方式使用5毫克mL -1的 BSA在步骤2.5中所述的附件。 </li><li>新增150分胶原蛋白肽，并允许它连接通过45分钟的myc基因标记的抗体。 </li><li>洗去多余的胶原蛋白，用PBS缓冲液。 </li><li>加入链霉亲和涂层的超顺磁性珠（珠1微米= 2微克mL -1和2.8微米微珠=毫升20微克-1），并允许它们附加到45分钟的胶原蛋白分子。 1微米的珠应在PBS稀释至所需浓度。 3微米微珠应分开使用强大的磁铁，然后在PBS resolubilized的解决方案。这个过程应重复3次。我们注意到，这一步是必不可少的GE牛逼的3微米微珠表面固定的胶原蛋白肽结合。 </li></ol> 4。部队蛋白水解实验<ol><li>一旦流动细胞组装与胶原蛋白肽和磁珠，在磁阱下低，〜1 PN力流细胞的图像。在我们的经验，亚珠有时弱坚持在一个非特定的方式表面。应用的微薄之力，确保这些珠子是解放。 </li><li>添加酶的激活流动池。在这种情况下MMP-1的预先激活，通过增加3.5毫米APMA（4-aminophenylmercuric的醋酸），并在37°C孵育3小时。使用SDS-PAGE电泳，激活验证。 </li><li>只要流动细胞的磁性镊子设备重新推出，记录视频跨越几个领域的观点，通常会产生数百附加珠。这将是T = 0。 </li><li>重复同一过程的记录来看几个领域（返回ţ○同一领域的观点在固定的时间点），直到所有的磁珠分离或没有进一步的蛋白质，是有迹可寻的。 </li><li>动力学数据分析<ol><li>珠计算在各个领域的角度来看，在不同的时间点和平均。正常化珠数珠珠在t = 0的平均数除以各时间点。这将确保我们可以比较来自不同实验的蛋白质率，因为珠的绝对数量将有所不同，从实验到实验。 </li><li>作为时间的函数绘制的比例。在这种情况下，我们安装了指数衰减，加上一个常数。 </li></ol><img alt="公式（3）" src="/files/ftp_upload/3520/3520eq3.jpg" /> ƒ（T）是附加（或胶原分子unproteolyzed），珠率，t是时间，是由胶原蛋白系绳连接珠子的分数，B衰变速率常数， 和 c是珠是连接非特异性的一小部分。 <ol start="3"><li>重复同样的实验在不同的力量和阐明MMP-1 MMP-1浓度的胶原蛋白水解的影响力。 </li></ol></li></ol> 5。代表结果上述协议描述了一个用于研究的酶水解的影响力的新型磁性镊子（ 图1）使用。我们校准镊子为1微米，3微米微珠使用所观察到的布朗波动的幅度和滚降频率的计算，在不同的磁铁位置（ 图2）。力水解实验中，设置类似的是，除了DNA的胶原蛋白（ 图3，4）取代。规格化数剩余珠可以绘制时间找到水解率（ 图5）的功能，而这个过程可以重复不同的酶的浓度和力量。 <img alt="图1" src="/files/ftp_upload/3520/3520fig1.jpg" /> 图1。磁阱校准过程示意图（未按比例）。两个常任理事国的稀土磁铁产生的磁场对超顺磁性珠拉。翻译磁铁向上和向下调整的作用力。珠通过两个磁铁之间的针孔的光线通过使用传统的亮场显微镜成像插图 ：图片采取与一个40X空中目标。尖锐，圆点对应连接到盖玻片表面的珠子。出的重点对象是独立的珠子。 <img alt="图2" src="/files/ftp_upload/3520/3520fig2.jpg" /> 从样品表面的功能作为一个磁铁的距离为1微米（ 左 ）和2.8微米（ 右 ）珠校准力图图2。第数据拟合经验功能<img alt="equation4" src="/files/ftp_upload/3520/3520eq4.jpg" /> ， 其中 x是从磁铁的距离。= 31.8，B = 5.61，C = 1微米微珠和4.39 = 140，B = 3.10和c = 1.86为3微米微珠。这些价值观是具体到特定的仪器和实验几何，每台仪器应单独校准。在每一个点的误差棒代表上珠，以珠为基础的作用力〜10％的变异，由于粒径的变化。施加的力是珠量成正比，珠体积变化超过平均〜9％（根据制造商的规格）。 <img alt="图3" src="/files/ftp_upload/3520/3520fig3.jpg" /> 图3。部队蛋白质检测设置（ 不按比例 ）。 通过 M盖玻片表面的胶原蛋白模型的三聚体YC /抗-myc基因的共轭。链霉亲和涂层的超顺磁性珠连接到胶原蛋白的三聚体通过生物素 - 亲和联动。活化中MMP-1的削减胶原蛋白随着时间的推移，造成珠脱离表面和移动焦平面。 <img alt="图4" src="/files/ftp_upload/3520/3520fig4.jpg" /> 图4。示意图卡通蛋白质作为时间的函数。卡通显示发生的观点随着时间的推移蛋白质样品领域。随着时间的推移，MMP-1切胶原蛋白和磁珠分离和磁场的影响下，从焦平面移动。 <img alt="图5" src="/files/ftp_upload/3520/3520fig5.jpg" /> 图5。蛋白水解率取决于作用力16。所示的1.0 PN（3μM的MMP-1的， 黑色 ）收集的数据，PN PN 6.2（3μM的MMP-1的， 红色 ）和13（0.2微米MMP-1的; 法师NTA）。蛋白质的利率（拟合参数）：0.22±0.02分钟-1（1 PN），0.46±0.09分钟-1（6.2 PN），2.08±0.18分钟-1（13 PN）。在很长的时间点（15分钟）unproteolyzed珠的分数仍约在0.25〜在不同的实验常数。错误酒吧对应的泊松统计反映在每​​个时间点的若干意见。在每个时间点n 的珠子错误是 n 1/2。附加珠分数的错误，计算错误propagation.1微米微珠用于PN 1.0和6.2 PN 13 PN实验实验和2.8微米微珠使用。

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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=Mechanoenzymatic+cleavage+of+the+ultralarge+vascular+protein+von+Willebrand+factor.">Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor.</a> Science. 324, 1330-1334 (2009).</li><li>Woodside, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 6190-6195 (2006).</li><li>del Rio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+single+talin+rod+molecules+activates+vinculin+binding.">Stretching single talin rod molecules activates vinculin binding.</a> Science. 323, 638-641 (2009).</li><li>Gosse, C., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers:+micromanipulation+and+force+measurement+at+the+molecular+level.">Magnetic tweezers: micromanipulation and force measurement at the molecular level.</a> Biophys. J. 82, 3314-3329 (2002).</li></ol>

没有利益冲突的声明。

本协议描述了一个经典的单分子技术的新用途。磁性镊子允许介质在成本效益的方式，以高通量单分子检测。然而，像所有的实验技术，也有挑战和潜在的隐患。 磁性镊子的限制磁光阱相比，一吨设备的空间和时间分辨率低。此外，这里描述的简单吨，产生的力是30 PN或以下，明显比AFM实验中经常访问的力量。这种限制可以是一种美德：吨是适合申请子-PN?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>微盖玻璃＃1.5（22X22） </td><td>厂商VWR </td><td> 48366-067 </td></tr><tr><td>微盖玻璃＃1.5（22x40） </td><td>厂商VWR </td><td> 48393-048 </td></tr><tr><td>噬菌体DNA </td><td> Invitrogen公司</td><td> 25250-010 </td></tr><tr><td> T4 DNA连接酶</td><td> Invitrogen公司</td><td> 15224-041 </td></tr><tr><td>单片机Ultracel YM-100 </td><td> Millipore公司</td><td> 42413 </td></tr><tr><td>抗地高辛</td><td>罗氏诊断</td><td> 11-333-089-001 </td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P9416-100ML </td></tr&gt; <tr><td>抗-myc基因抗体</td><td> Invitrogen公司</td><td> 46-0603 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> B4287-5G </td></tr><tr><td>磁珠的M-280链霉</td><td> Invitrogen公司</td><td> 658.01D </td></tr><tr><td>磁珠MyOne T1的链霉</td><td> Invitrogen公司</td><td> 658.01D </td></tr><tr><td>醋酸P-Aminophenylmercuric </td><td> Calbiochem公司</td><td> 164610 </td></tr><tr><td>生物素 - 马来酰亚胺</td><td> Sigma Aldrich公司</td><td> B1267 </td></tr><tr><td>生物素标记的寡核苷酸</td><td>内幕交易审裁处的DNA </td><td>定制合成</td></tr><tr><td>地高辛标记寡核苷酸</td><td>内幕交易审裁处的DNA </td><td>定制合成</td></tr><tr><td>胶原肽基因</td><td> DNA的2.0 </td><td>定制合成</td></tr><tr><td>中MMP-1基因</td><td>哈佛质粒数据库</td><td></td></tr><tr><td> Z-翻译</td><td> thorlabs </td><td> MTS50 </td></tr><tr><td>伺服控制器的翻译</td><td> thorlabs </td><td> TDC001 </td></tr></tbody></table>

multiplexed single-molecule force proteolysis measurements using magnetic tweezers

机械力的生成和检测是一个无处不在的细胞生理学方面，直接关系到癌转移1，动脉粥样硬化和伤口愈合3。在这些例子中，每个细胞都在他们的周围施加力量，同时酶解改造外基质（ECM）。流脑的力量的作用也因此成为一个面积相当大的兴趣，由于其可能的生物学和医学的重要性4-7。 如光阱，原子力显微镜，磁镊子10,11单分子技术使研究人员能够探测功能的酶在分子水平上对个别蛋白质的力量发挥。这些技术，磁镊子（吨），其低成本和高吞吐量显着。吨在〜1-100 PN的范围施加的力量，可以提供毫秒级的时间分辨率，12在单分子水平研究酶机制很好地匹配的素质。在这里，我们报告一个高度并行吨的实验研究单个蛋白质分子的蛋白质的影响力。我们提出具体的例子，由基质金属蛋白酶1（MMP-1）的三聚体胶原蛋白肽的蛋白质，但是，此法可以很容易地适应学习其他基板和蛋白酶。

Watch this Scientific Journal Video about Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers at JoVE.com

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

在这篇文章中，我们描述了利用磁性镊子，在一个高度并行的方式在单分子水平研究的酶水解的影响力。

警队复用单分子蛋白水解测量磁性镊子

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

multiplexed-single-molecule-force-proteolysis-measurements-using

Research

JoVE Journal

Bioengineering

11.5K 次观看.  Stanford University. 在这篇文章中，我们描述了利用磁性镊子，在一个高度并行的方式在单分子水平研究的酶水解的影响力。

视频: Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

伸展的DNA短序列恒力轴向光镊

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

科研

生物工程

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

使用滑翔分析生物大分子的抗弯刚度测量

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

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

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

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.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

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

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

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

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

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

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

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

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

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

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

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

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

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

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

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

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

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.

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.

使用荧光显微镜在剪切流下描述单分子构象变化

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