这项工作是由卫生部授予GM094246，美国心脏协会授予11GRNT7890004，以及美国国家科学基金会资助的MCB-1110501国家机构的支持。

1，创建构建含利息的蛋白质克隆表达目的蛋白到合适的载体的基因。使用载体如pcDNA系列或pRK5，因为它们非常适合用于在哺乳动物系统，如HEK293和CHO细胞中表达。 2，选择网站上的蛋白与荧光基团进行标记<ol><li>选择标签的网站，都能够体现出蛋白质中可能的构象变化。如果可能的话，使用同源蛋白的蛋白质或晶体结构，以帮助做出此决定。如果没有晶体结构信息，可以使用在线软件来预测蛋白质的可能的结构，从而获得洞察适当部位。 </li><li>选择的残基，使得所选择的残基的侧链是表面暴露的和可访问的荧光团，以使蛋白可以被标记。 <ol><li>选择残渣不是关键的，以蛋白质的功能，对于未参与配体结合位点的例子。 </li><li>在引入半胱氨酸突变，会优先考虑网站是类似的，例如丝氨酸，会导致极小扰动的蛋白质结构。 </li><li>选择标记位点后，进行突变，以确保该蛋白仅给出LRET从这些目的站点。使用标准的定点诱变方案产生突变，远离非二硫键连接的半胱氨酸可能潜在地结合成马来酰亚胺缀合荧光标记。不发生变异程二硫键连接的半胱氨酸和掩埋，游离的半胱氨酸，因为它们不应与在蛋白质的折叠形式的荧光染料反应。 </li></ol></li><li>通过使用标准的定点诱变协议点突变引入的半胱氨酸残基在所需的位点。 </li><li>包括蛋白酶切割位点可以特异性裂解掉从蛋白质中的半胱氨酸中的一个。如果蛋白质序列允许它通过保守突变蛋白质有凝血酶（识别序列LVPRGS）或引入网站Xa因子（识别序列IDGR或IEGR）附近引入的半胱氨酸序列和访问蛋白酶裂解;否则，四或六肽序列可被插入作为一个整体。选择的部位，使得在切割时所引入的半胱氨酸残基中的一个解离的其余部分蛋白在某些情况下，这可能需要两个切割位点侧翼的突变的半胱氨酸。 </li></ol> 3，测试蛋白的表达和功能<ol><li>进行免疫印迹确认突变蛋白的表达。 </li><li>执行该蛋白质的功能测定法，以保证所述突变改变了蛋白质的功能仅最低限度，如果在所有，以避免研究一种不正常的蛋白质的构象变化。 注：由于所有的蛋白质具有不同的功能，也没有一架F<html> unctional测定，是专门用于LRET;然而，功能测定法的一些实例包括酶的活性测定法的酶，配体结合测定法对受体和电生理研究离子通道。 </li></ol> 4，选择荧光基团被使用基于预期的距离范围选择荧光基团被测量，使得所述范围是0.5-1x之间的荧光团对中的R 0。 注意：这允许为背景，其典型地具有更长的寿命的一个更简单的减法。例如，如果正在测量的预期距离范围大约是35埃，一个合适的荧光团对使用将是铽螯合物作为供体和Alexa 594作为受体，因为R 0为这对是53埃（ 表1）。 5，通过瞬时转染哺乳动物细胞所需的量表达蛋白ntent“&gt;瞬时转染的兴趣到使用任何常见的转染试剂的选择哺乳动物细胞中的蛋白质通常情况下，使用每LRET实验4个10-cm的培养皿为HEK和CHO细胞系，然而，该量可以根据蛋白质表达变化性，稳定性等 ，允许细胞表达的蛋白质为收获前36-48小时。 6标签的蛋白质<ol><li>分离从培养皿的细胞。通过简单的移液缓冲对盘底部分离HEK细胞。分离的CHO细胞用细胞刮刀，洗去带外缓冲介质。 </li><li>收集细胞，离心，在1100×g离心3分钟。使用这些相同的离心分离机的设置标签后收集细胞，以及随后的洗涤之后。 </li><li>暂停细胞在3ml胞缓冲液中，然后加入等摩尔量的供体和受体荧光团高达100-300纳米的最终浓度。孵育在肩˚F或1小时，在室温下。 </li><li>洗3-4倍带外缓冲器以除去未结合的荧光团，然后暂停这些细胞在胞外缓冲液（通常为2毫升）LRET测量这些标记的细胞。 </li><li>作为对照，标签一组单独的细胞，只有供体荧光团（铽螯合物）中不添加任何受体荧光基团。 注：从这些捐助者只实验数据要完整的分析。这些实验可以在相同或不同的日子进行。 </li><li>再次，进行功能验证，此时用标记的突变体蛋白，如标号处理，也干扰功能取决于用于标记的部位。 请注意：这些实验可以在相同或不同的日子进行。 </li></ol> 7，搭建实验LRET <ol><li>将重悬的细胞中的石英比色皿与1毫升的最小体积。 </li><li>打开计算机和仪器，并调整DAT的参数à收购方案相应。 <ol><li>将激发波长为供体荧光团（330-340纳米的铽螯合物效果很好）的吸收范围。 </li><li>设置适当的发射波长，同时考虑到受体的排放变化的基础上所使用的受体。重要的是，选择尺寸仅为受体排放，不包括任何流血，通过从捐赠者的排放检测波长。例如，使用565纳米的Alexa的555波长作为受体表1。对于捐赠者只测量，其中蛋白标记只能与捐助，但没有接受，使用545纳米波长测量铽螯合物的排放。 </li><li>设定排放检测的长度至少为3倍的预期寿命LRET以确保长寿命组件将不会被错过。 </li></ol></li><li>执行扫描。做99次扫描至少三次扫描，以确保结果的一致性。除日Ë结果为文本文件（*。TXT）。 </li><li>来测量蛋白质对于不同的条件（如增加的配体）的构象变化，改变这些条件，并再次这个新的条件下对同一样品执行的每99次扫描的至少三个扫描。如果学习上的谷氨酸受体的构象谷氨酸的效果，例如，添加谷氨酸至1mM到扫描在步骤7.3中相同的样品，然后再次扫描。 </li><li>最多可添加五个单位适当的蛋白酶，并采取持续扫描，直到分裂完毕，并没有进一步的变化被认为是在生命周期的三个连续扫描。通常情况下，切割完成的两到3小时。如果Xa因子切割位点被用于蛋白酶裂解，例如，添加3μl的Xa因子的样品，并进行扫描，每30分钟3小时。 </li></ol> 8，分析得到的数据<ol><li>打开数据分析软件。 </li><li>装载荧光寿命通过输入ASCII打开文本文件中的数据。加载所有的重复以及最后的背景测量，合并成一个文件。 </li><li>平均从所有扫描的数据为各实验条件下，以得到最终的数据。要做到这一点，强调的是包含各试验中的列，然后在菜单栏中的数据菜单上单击统计上排显示从试验的平均荧光强度，以及标准差和标准差。 </li><li>积的平均值作为线图上的Y轴和寿命中的X轴，以创建表示该受体的敏化发射的寿命的曲线微秒荧光强度。改变剧情的Y轴为对数刻度，以便更好地可视化数据。 </li><li>在背景上的测量数据，重复步骤8.3，平均，显示重叠显示完全解理最终扫描。 </li><li>显示的平均背景资料相同的情节包含平均的原始数据。要做到这一点，在打开的绘图菜单中的图层控制对话框。然后，将含有背景数据传输意味着成层的内容的列表。 </li><li>对齐的平均背景数据的平均的原始数据。要做到这一点，使用在数学菜单中简单的数学函数相乘，或根据需要，直到尾部的背景与尾部的原始数据重叠划分的背景资料。 注：在此尾端，从感兴趣的蛋白质的寿命应该已经完全消失了;简单的背景信号存在什么对齐是之前和蛋白酶切割后。 </li><li>从最初的原材料LRET信号中减去对准背景信号，用简单的数学函数了。 </li><li>拟合数据以指数衰减（单个或多个指数函数，根据不同的位点和实验设计）。 <ol><li>设置起始边界拟合开始结束之后的激光脉冲。使用同样的起始点为所有后续的曲线配件。 </li><li>在选择拟合函数对话框中，选择指数衰减，以适应终身下面的等式为单一指数衰减： <img alt="式（4）" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq4.jpg" width="200" />式。 4 其中，y表示荧光强度，Y 0表示背景强度由于从系统噪声中，A 1是信号强度，t为荧光寿命，x为时间，且x 0是时间偏移量。 </li><li>修正X 0比0，开始拟合函数，并拟合数据。 注：如果实验设计只从一个对网站有信号时，所得到的数据应该很容易适合由一个单一的指数衰减8。拟合的残差是一个很好的方法来确定的拟合优度，如果额外的衰减寿命是requir主编。 </li></ol></li><li>使用从数据获得的寿命来计算使用的Forster方程（方程1和2以上的重排）的荧光团之间的距离： <img alt="式（5）" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq5.jpg" width="200" />式。 5 注意：所有的变量如上面提到的与τDA已被测定为致敏受发光寿命。在R 0和R的测量进一步的细节和例子可以在其他地方7,9,10中找到。 </li><li>重复实验和数据分析至少三次，以确保重现性。同一测量个体之间的重复矛盾提出了质疑数据的有效性;使用额外的控制实验或重复。计算使用免费古斯塔夫误差分析计算器（博士托马斯·胡伯开发）或类似的系统，传播错误的测量误差适合生存期。 </li></ol>

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作者什么都没有透露。

LRET是一种强大的技术，使科学家可以在一个单一的蛋白质，以及在多聚蛋白亚基之间测量域之间的距离。正因为如此，LRET是非常适合于研究的蛋白质或其他大分子的构象变化和动力学。上述协议应允许适当装备的实验室能够轻松地测试他们的假设;然而，也有错误的可能困扰新调查许多常见来源。如果很少或没有LRET信号看出，首先检查所用的波长设置。激励应铽（330-340纳米）的吸收范围。为施?...

荧光共振能量转移（LRET）是著名的荧光共振能量转移（FRET）技术1的衍生物。到FRET相似，LRET可用于测量距离和附着在上的10-100埃1-3的范围内的感兴趣的蛋白的特定位点供体和受体荧光团之间的距离的变化。是LRET的原理也类似于在该共振能量转移到FRET发生2近侧的荧光团之间时，供体荧光团的发射光谱与受体荧光团的吸收光谱重叠。这个转移的效率有关，这两个荧光团由下面的等式之间的距离： <img alt="式（1）" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq1.jpg" width="120" />式。 1 其中，R是两个荧光团之间的距离，E是连接的效率ERGY转移，且R 0，下面所讨论的，是福斯特半径为荧光团对， 即在其中传递的效率是半最大的距离。从这个等式中，可以看出，效率与上升到逆第六电源1的距离的大小。正是这种逆第六功率的依赖，允许FRET和LRET测量是连到小的距离的变化极其敏感，当FRET对的R 0附近。专门标记的蛋白质或其他大分子所需站点的能力允许我们利用这个敏感的优势，监控的构象变化。 时相比，荧光共振能量转移，其使用传统的有机染料分子，LRET提供了额外的优点。而不是使用有机染料作为供体荧光团，镧系阳离子，通常是铽或铕在LRET，用于1,4-6。那年秋天ü荧光升气管这一类， 如铽螯合物，也非常灵活的，因为它们可以具有广泛的受体荧光团的使用。这种灵活性是可能的，因为螯合的镧系元素的发射光谱包含多个尖锐的发射峰，允许一个单一物种供体荧光团能够与各种各样受体荧光团中的一个使用。因此，可以在没有任何担心从供体发射5污染泄流通过的被检测致敏的受体发射。实验者选择基于两个荧光团（ 图1和表1）之间的期望距离的特定受体。在这些螯合的镧系元素的荧光团，该金属离子是由包含敏化的通常较差吸收的镧系元素的激发以及丰富的生物反应性官能团的系绳的离子对特定官能团的高分子1的天线组分子螯合， 5,6。 ONCê兴奋，镧系元素，通过光子带衰减率在毫秒范围内释放放宽到基态。由于衰变既不是单峰对单峰松弛也不三重态到单重态放松，光子的发射不能适当地被称为荧光或磷光，而是更恰当地称为发光1。稀土发光的长期​​衰退极大地有助于寿命测量。寿命的测量可以被用来确定通过下列关系效率： <img alt="式（2）" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq2.jpg" width="120" />式。 2 其中，E为转移的效率，τD是供体（螯合稀土）的时候不参与能量转移的寿命，τDA是供体的寿命与受体能量转移，当参与。随着LRET，τDA可以人所以被测量作为致敏受体发射的寿命，因为铽的寿命是这样比的有机受体荧光团大得多。具有相同的寿命作为其指使激发（供体的镧系元素）的受体发射，并从受自身的固有荧光寿命的寿命的任何贡献相对可以忽略不计。通过测定致敏发射，而不是供体发射，我们也无需确保标签在恰好为1：1的比例供体与受体。蛋白质可以改为同时与受体和供体荧光标记。 à不均匀标记人口造成的，但双供体标记的蛋白质将不会发出在接受波长和双受体标记的蛋白质将不激动。此外，荧光团之间的距离应该是相同的，而不管其中的半胱氨酸位点的特定荧光团附着，使用各向同性的镧系元素作为供体尤其是当，所以娘家姓d，来指定一个特定的网站分别接受供体或受体是不必要的。强度可能会受到与异质群体，但仍应该超过足以被检测到。 在规划的实验中，荧光团的选择应是R 0值的一对，以及预期的距离范围来规定被测量。在R 0的值被定义由下面的等式： <img alt="式（3）" fo:content-width="2.5in" src="/files/ftp_upload/51895/51895eq3.jpg" width="250" />式。 3 其中，R 0为福斯特半径埃，κ2是两种染料（通常假设为三分之二）之间的取向因子，φD为供体的量子产率，J为光谱重叠积分捐赠者的间发射光谱和受体的吸收光谱中的M - 1-1处4，且n是介质1的折射率。 我们的实验室已通过对蛋白质的供体和受体标记位点之间引入蛋白酶识别位点被探测添加修改常规LRET技术。该变形允许调查在未纯化系统，例如整个哺乳动物细胞7。使用半胱氨酸作为位点标记时，由于在与结合于半胱氨酸的巯基，其它蛋白质上具有半胱氨酸还标记了的细胞的马来酰亚胺共轭染料标记的过程中该技术是特别有用的。然而，通过包括对感兴趣的蛋白的蛋白酶裂解位点和之前和裂解后测量的寿命，实验者可以定量地减去从原始信号蛋白酶裂解后的背景信号。这个减法隔离感兴趣的蛋白质产生的特定信号（ 图尤尔2）。使用上述的变形例中，LRET可用于测量铽螯合物供体和对蛋白质的受体探针之间的距离的变化，并由此监视未经纯化的要求，在蛋白质的接近生理状态的构象变化。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig1highres.jpg" width="500" /> 图1，吸收和螯合铽在黑，以及作为代表性受体的Alexa 488，在红色的发射光谱图。注意，多个发射峰和铽螯合物的各峰的尖锐，窄的发射范围。这种模式允许对与多种受体荧光团的用铽和便于这些范围，其中铽表明没有发射内致敏发射的测量。在486铽的发光峰值波长与A重叠相当不错<html>的Alexa 488的bsorption峰，允许共振能量转移两个荧光团之间发生。 515nm的波长是一个很好的选择，检测致敏排放为这双，因为它是在铽发射峰之间的山谷中，相当接近520纳米的Alexa 488的发射峰。注意，被附近的受体峰，虽然理想的，则不需要-565（nm）是仍然能够检测到没有同时检测铽发射的Alexa 488发射。 <table border="0" cellpadding="0" cellspacing="0" width="374"><tbody><tr height="27"><td height="27" style="height:27px;width:147px;"> 受体荧光基团 </td><td style="width:45px;"> R 0（） </td><td style="width:183px;"> 发射波长（nm） </td></tr><tr height="23"><td height="23" style="height:23px;width:147px;">阿托465 </td><td align="right" style="width:45px;"> 36 </td>3px的;“&gt; 508 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;">荧光素</td><td align="right" style="width:45px;"> 45 </td><td align="right" style="width:183px;"> 515 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的488 </td><td align="right" style="width:45px;"> 46 </td><td align="right" style="width:183px;"> 515 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的680 </td><td align="right" style="width:45px;"> 52 </td><td align="right" style="width:183px;"> 700 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的594 </td><td align="right" style="width:45px;"> 53 </td><td align="right" style="width:183px;"> 630 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的555 </td><td align="right" style="width:45px;"> 65 </td><td align="right" style="width:183px;"> 565 </td></tr><tr高度=“22”&gt; <td height="22" style="height:22px;width:147px;"> Cy3标记</td><td align="right" style="width:45px;"> 65 </td><td align="right" style="width:183px;"> 575 </td></tr></tbody></table> 表1常用的受体荧光基团用铽螯合物作为捐助11 LRET列表。在R 0的值进行测定时，供体和受体被装AMPA受体的可溶性受体激动剂结合域。它是理想的，重新测量R 0值正在研究每一个新的系统。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig2highres.jpg" width="500" /> 图2呈现的LRET方法的概述。（A）中的 AMPA受体是一种膜蛋白，其经历在配体结合构象变化。在翻盖型配体的双nding域在这里红色圆圈。 （B）存在于开放构象（左）AMPA的在未结合蛋白的配体结合结构域。当绑定到配体谷氨酸，在蛋白质周围的配体（右）关闭。通过将荧光团在证明的站点上的发光二极管，此构象变化的性质，可以被看作是荧光团的改变，这将影响随后的荧光寿命之间的距离。 （C）当标记的全细胞，可能会发生的利息两者蛋白质以及背景的膜蛋白的标记（左）。蛋白酶裂解后，从感兴趣的蛋白质LRET信号就会消失，由于可溶性片段的释放，而使背景信号完好（右）。此背景信号然后可减去从原始信号。

<table border="0" cellpadding="0" cellspacing="0" width="1394">
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:693px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">LanthaScreen Thiol Reactive Tb Chelate</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">PV3579</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Fluorescein-5-Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">F-150</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 488 C5 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-10254</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 555 C2 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-20346</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 594 C5 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-10256</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acceptor fluorophore-Alexa Fluor 680 C2 Maleimide</td>
			<td style="width:223px;">Life Technologies&#160;</td>
			<td style="width:119px;">A-20344</td>
			<td>Choice of acceptor depends on the specific experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">QuantaMaster 3-SS</td>
			<td style="width:223px;">Photon Technology International</td>
			<td style="width:119px;"></td>
			<td>Spectrofluorometer should have pulsed excitation with the ability to measure lifetimes in the ms range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FluoreScan 2.0</td>
			<td style="width:223px;">Photon Technology International</td>
			<td style="width:119px;"></td>
			<td>Data Acquisition Software used in manuscript. Software provided with fluorescence instrument.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Origin 8.6</td>
			<td style="width:223px;">OriginLab</td>
			<td style="width:119px;"></td>
			<td>Origin is the data analysis software used in protocol. Can use other similar data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Quartz Cuvette</td>
			<td style="width:223px;">Starna Cells, Inc</td>
			<td style="width:119px;">3-Q-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Stir Bar&#160;</td>
			<td style="width:223px;">Bel-Art Products</td>
			<td style="width:119px;">F37119-0007</td>
			<td>Used in cuvette to keep cells in suspension. Can use any stir bar that fits the cuvette</td>
		</tr>
	</tbody>
</table>

luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells

发光共振能量转移，或LRET，是用来测量在10-100埃的距离范围内的蛋白质两个站点之间的距离的功能强大的技术。通过测量各个连接的情况下的距离，该蛋白质的构象变化可以很容易地进行评估。与LRET，镧系元素，最多螯合铽，用作供体荧光团，得到的优点，​​如更长的供体 - 仅发光寿命，使用多个受体荧光团的灵活性，并有机会检测致敏受体发射作为一种简单的方法测量不也检测供体只有信号的风险能量转移。在这里，我们描述了使用LRET上表达，并测定完整的哺乳动物细胞的表面上的膜蛋白的方法。我们介绍了荧光LRET对之间蛋白酶切割位点。获取原始LRET信号后，乳沟在该网站将删除的蛋白的特异性信号LRET息使我们能够定量地减去该保持切割后​​的背景信号。此方法允许更要作出生理学相关的测量，而不需要蛋白质的纯化。

一个成功的LRET测量与镧系捐赠者应该有一个捐赠者只在毫秒级的时间范围内的寿命。致敏LRET排放的标有两个供体和受体的蛋白质的寿命会明显缩短，与后背景减除图3微秒范围内的寿命。蛋白酶裂解导致增加的寿命应该成为稳定一段时间（ 即不再改变），这表明蛋白酶切割完成图4，如果LRET信号来自只有一组位点，背景减去后所得到的发光寿命应该给单指数寿命。...

Watch this Scientific Journal Video about Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells at JoVE.com

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

我们在这里介绍了一种改进荧光共振能量转移（LRET）方法，在这里我们介绍的供体和受体荧光基团的站点之间的蛋白酶裂解位点。该变形使得我们能够获得来自感兴趣膜蛋白，使膜蛋白的无蛋白质纯化的研究中出现的具体LRET信号。

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

luminescence-resonance-energy-transfer-to-study-conformational

Research

JoVE Journal

Bioengineering

11.9K 次观看.  University of Texas Health Science Center at Houston. 我们在这里介绍了一种改进荧光共振能量转移（LRET）方法，在这里我们介绍的供体和受体荧光基团的站点之间的蛋白酶裂解位点。该变形使得我们能够获得来自感兴趣膜蛋白，使膜蛋白的无蛋白质纯化的研究中出现的具体LRET信号。 

视频: Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

负染色的EM可视化蛋白质和大分子复合物：从电网的制备图像采集

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

科研

生物工程

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

在胎儿肺细胞的机械传导的实验系统研究

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

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

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.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...