This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1.脂质制备<ol><li>分装所需量的脂质成透明玻璃试管中。利用E.大肠杆菌极性脂质提取物，但大部分脂质组合物都可以使用。如果脂质是粉末形式，悬浮它们的氯仿至20毫克/毫升的浓度。干燥的脂质在室温下N 2气，直到所有溶剂蒸发。 </li><li>悬浮在戊烷的脂类，再晾干气体N 2源源不断除去氯仿遗留的痕迹。干，直到所有溶剂蒸发。而干燥，轻轻旋转管，使得所述脂质分布在均匀的膜覆盖所述管的底部第三而非形成致密质在底部。 </li><li>添加含洗涤剂重建缓冲液（300mM的氯化钾，50mM的柠檬酸，25mM的K 2 HPO 4，pH为4.5，35毫CHAPS）溶解脂质。使用其他温和的清洁剂进行这一步，如果蛋白质是很差容忍CHAPS。 </li><li> ResuspenD使用水浴超声波仪中的脂质对清晰度。浸入管在水中超声发生器浴中的最大功率的中心在短（30秒-1分钟）脉冲短（30秒-1分钟）暂停。该解决方案应该变得清晰。 注：清晰的最终程度实现取决于所使用的特定的脂质组合物。一些批与批变异在该步骤中，甚至在名义上相同脂质，也是可能的。残留雾度是由于光散射的大颗粒在悬浮液中，这可通过离心除去。 </li><li>在室温下孵育的悬浮脂类为20-60分钟。 </li></ol> 2.脂蛋白体的形成注：几种策略可以用来插入洗涤剂增溶的蛋白质到脂质体中。对于CLC-EC1透析效果很好，因此是选择6,9,10的方法。 <ol><li>添加纯化的蛋白质的期望的蛋白质与脂质（P / L）的比值（表示为微克Ò<html> F蛋白/毫克脂质）。的P / L的比例的选择取决于实验的目的。 <ol><li>如果目标是评估所关注的蛋白质的活性，重组在高的P / L的最大化信号，因为每个脂质体含有的蛋白质的多个副本。量化的活性和/或蛋白的单一传输速率，重建在泊松稀释制度低的P / L的，以使每个小泡平均含有1蛋白质。见第7步和讨论进行了较深入的分析时，使用每个政权。 </li></ol></li><li>来确定用于各种治疗方案的最佳P / L的值，执行活动的完整滴定作为蛋白质浓度8,11-13的函数。然而，对于激活单元中的任何蛋白质的量，分子量（MW，以kDa表示）被称为后的P / L的值可以被用作初始瞎猜： 皮克“/&gt; <img alt="公式2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li>在预润湿的透析管或盒加载的蛋白质和脂质的混合物。将透析设备在含有500-1,000x脂质重建缓冲液的体积的烧杯中（ 即 ，进行1 ml类脂再悬浮的1升缓冲液）下缓慢搅拌。 <ol><li>改变缓冲液的3-4倍的间隔&gt; 3小时。在每一个解决方案的变化，洗磁带/管和用蒸馏水的烧杯中，注入新鲜的重建缓冲液的烧杯中。包括1-2 O / N区间的解决方案转变。确切的数目的溶液的变化和持续时间取决于所用的确切的脂质和去污剂。 </li></ol></li><li>在最后一步完成后，从透析器去除脂质体，分装在其中管和闪光灯在-80℃冻结，在液体N 2或冻结。 </li></ol> 3.记录的建立 &lt;P类=“jove_content”&gt;注意：记录设置（ 图2A）包括两个腔室（平底缸，〜3-4毫升容积）中，Cl -的（见下文）的电极，pH计用模拟或数字电输出，数字转换器，和一个计算机用适当的采集软件。 <ol><li>连接Cl -的电极pH计，pH计，以被连接到计算机的数字化板的输出。放置在一个腔室中的参考电极和重新编码导线中的另一个（ 图2B）。 注：导线的极性是正确的，如果ΔV 校准 （见步骤6.4和7.2）是正的。 </li><li>放置室上的轰动板录音室（ 图1B）的搅拌棒。确保搅拌棒不接触电极。 </li><li>连接使用琼脂桥（ 图2B）（100毫米氯化钾和2％琼脂糖）的腔室。存储在100毫米氯化钾琼脂桥梁。 </LI&gt; </ol> 4.制备的Cl -电极<ol><li>以一个老-possibly-非正常pH电极。打破玻璃涂层完全显示出来的银线。 </li><li>小心地从使用手术刀刀片的银线去除涂层。清洗用H 2 O和乙醇的丰富金额的电线。 </li><li>发生的FeCl 3（或漂白剂），直到导线的饱和溶液覆盖有均匀的深色涂层氯化银的。 </li></ol> 5.准备单层囊中<ol><li>夜的实验之前，膨胀将1g的Sephadex G-50珠在15ml外部缓冲液（1mM的氯化钾，150mM的K 2 SO 4，25mM的柠檬酸，pH4.5）中，轻轻摇动（不搅拌），用于至少3个小时，在室温后再使用。每个实验需要〜3个毫升肿珠。保持溶胀的珠粒于4℃几天至多。 </li><li>而脂质体解冻在室温，倒在每列〜3米升肿G-50珠。让他们干通过在RT重力流;这通常需要1-2分钟。 </li><li>通过使用0.4米聚四氟乙烯截止通过Mini-挤出机挤出的脂蛋白11次准备单层囊。 </li><li>放置在一个塑料圆底管色谱柱。旋柱以除去过量的溶液。在临床离心机离心20-30秒在1400 XG。 </li><li>丢弃流过，地点列在一个13×100毫米的玻璃管中，并添加100微升挤出囊泡到柱上。在临床离心机离心柱1分钟，在500×g离心。 </li><li>收集〜200微升流动通过的，将被添加到记录室，在步骤6.5。 </li></ol> 6.流出测量<ol><li>放置加入2ml的100mM氯化钾在基准（接地）室和1.8毫升外部缓冲液（1mM的氯化钾，150mM的K 2 SO 4，25mM的柠檬酸，pH4.5）中，以记录室中。内部的细微渗透压失衡和外部的解决方案不影响</li><li>启动收购程序。让基本平衡，这可能需要几分钟的时间。 </li><li>一旦信号达到稳定的基线（脂质体是准备和列干），开始记录（ 图3A）。 </li><li>加入15微升10mM的KCl溶液校准系统（ 图2A）。 </li><li>添加脂质体。阿细跳可能是可见的，由于不完全拆除外的Cl -从脂蛋白（ 图3A）。等到基线稳定。 </li><li>加入缬氨霉素（1微升以1mg / ml，在EtOH中）来启动流出（ 图3A）。 </li><li>让流出的运行时间过程，直到它高原（ 图3A）。 </li><li>加入40微升在外部缓冲器以溶解所有的脂质体（ 图3A）中制备1.5M的β-辛基葡糖苷（β-OG）。结束录制。 </li></ol> 7.数据nalysis <ol><li>从ASCII或文本格式导出跟踪文件，并将其导入到所选择的分析程序。 </li><li>测量的电压在以下各点（ 图3A）： 在记录开始时（V 0）; 此外校准脉冲（V CAL）之后; 在加入脂质体（V 脂 ）的; 在流出（Ⅴ 翅片 ）的端部; 在加入洗衣粉（V TOT）的; 基线的电压使V 0 = 0。 </li><li>使用能斯特 - 普朗克公式确定的实验值<img alt="公式3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" />通过测量 <img alt="公式4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> 其中，卷校准是在校准脉冲在微升的体积，1800是腔室容积在开始的实验中表达1，L，[CL] 卡/中是氯-在毫米外部缓冲器的校准脉冲和浓度ΔVCAL的校准脉冲后测得的跳毫伏。 注：此经验确定α的有三个目的：它保证了系统正常响应，提供实验之间的仪器的响应的一致性的措施，以及检查是否有错误是在溶液中做出决策。 </li><li>每一步之后计算氯浓度如下： <img alt="公式5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="方程6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="公式7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> 该因子四百分之三来自稀释15微升校准脉冲的进入2000微升最终体积。 </li><li>演算吃在每一步，ΔCL 脂/鳍/ TOT的变化[氯]。 </li><li>转换V（T）的氯（T）与 <img alt="方程8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> 在临界点直接决定（CL）值，并将其进行比较，以计算出的值作为内部对照。 </li><li>标准化的跟踪，这样的氯相对 脂 = 0和Cl 相对 托特 = 1 <img alt="公式9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li>适合的流出时间过程，以下面的等式： <img alt="10式" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> 其中L是Cl的速率-漏是通过实验，通过测量确定的氯-从在相同条件下，τ制备无蛋白质的脂质体流出是过程的时间常数。 </li><li>计算V，初始速度： 其中，ΔCL 翅片表达于毫摩尔，V是在微升室的容积。 </li><li>计算运输率/电导率 <img alt="12式" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> 其中，p是在P / L时，MW为活性复合物的分子量以kDa表示</li></ol>

<ol>
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The authors declare no competing financial interests.

我们已经描述了详细的协议来测量Cl -的运输由纯化的阴离子选择性通道或转运蛋白重构于脂质体介导的。所使用的例子是原核H + / Cl -的换热器CLC-EC1。然而，该方法可以容易地适合于研究通过配体12,13,15，11,12的电压，或者通过更换银运动不同的阴离子选择性15,16门控通道：与一种适合于所考虑的离子氯化银电极。电极选择性离子比其它氯- ，如H <su...

在过去的二十年中，以过表达和纯化膜转运蛋白的能力显着增加：离子通道，初级和次级转运目前一般均从异源表达系统，以及天然来源纯化。新的方法来监测表达，改善和促进提取和提高这些蛋白质的稳定性正在不断开发1-5。这些技术的突破已经有助于触发的原子级的结构信息上的膜蛋白，这反过来，提高了我们自己的功能的结构基础的理解的爆炸。相比之下，我们的能力来探测纯化的蛋白质的功能性质没有增加以同样的速度，这样，在某些情况下，高分辨率的结构信息是伴随着定性功能数据，从而限制了我们的定量测试结构为基础的预测能力。因此，发展定量和可普及功能测定t是朝着膜蛋白功能的机械基础的澄清的关键步骤。 在这里，我们描述了可用于定量测定纯化和重组的Cl键的功能性质外排测定-通道和转运。该测定法的基本原则可以推广到各种运输系统，以及非离子转运蛋白。脂质体重组，纯化的Cl -的信道/转运在一个大的氯的存在下-梯度（ 图1A，B）。 Cl -的流出是通过加入一种离子载体的启动，以允许对离子通量，在我们的情况下，第K +离子载体缬氨霉素，其分流由氯建立的电压-梯度和设置初始膜电位至K的平衡电位+ 6,7。没有T他离子载体没有显著净Cl -的流出发生的，因为它防止了由一个跨膜电位的产生。 -流出，并且f 0，不含有活性蛋白的脂质体的分数τ，C1的时间常数：所述数据是由两个可测量参数（ 图1C）定量描述。从τ和f 0酉Cl -的传输速率，活性蛋白质的级分和活性复合体的分子质量可以衍生8。已知结构和功能的热交换器-该技术是利用脂蛋白重构与CLC-EC1，一个良好表征CLC型H + /氯这里示出。该测定是很容易推广到通道或转运与不同的离子选择性或其活性依赖于电压和/或配体的存在。另外，该测定可用于确定是否小分子直接影响重组蛋白，以定量这些化合物和如何膜组合物或脂质修饰试剂影响重构的通道和转运功能的影响。

<table border="0" cellpadding="0" cellspacing="0" width="995">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
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		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
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			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
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			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
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			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
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			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
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</table>

a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

我们描述了一个详细而强大的协议来衡量Cl -的运输由纯化CLC-EC1，原核CLC-H型+ /氯介导-交换，重组的脂质体。该实验的示意图示于图3脂蛋白体重组，纯化的CLC-EC1和含有高内氯-浸渍在含低氯浴溶液- 。在这些条件下的净Cl -的流出防止通过在脂质体（ 图3A）正电荷的累积。此外缬氨霉素允许K +而穿过膜从而分流的电?...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K 次观看.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

视频: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

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

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

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

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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

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

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

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

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...