We thank Prof. J. Stelling (Department of Biosystems Science and Engineering, ETH Zurich and Swiss Institute for Bioinformatics, Basel, Switzerland) for his critical review of the manuscript.

1.试剂准备<ol><li>以制备碳酸包被缓冲液，溶解0.36克Na 2 CO 3和0.84克的NaHCO 3在100毫升蒸馏水;无菌过滤器通过使用真空从动0.22μm的聚醚砜（PES）膜过滤器，并存储在RT之前使用的缓冲区。 </li><li>通过在磷酸盐中加入0.05％体积/体积的吐温20制备洗涤溶液缓冲盐水（PBS）。 </li><li>通过在100毫升PBS中5克BSA（≥98％），并存储溶解在4℃下制备的5％牛血清白蛋白（BSA）的PBS溶液（封闭溶液）。 </li><li>重组受体，配体和阻止肽<ol><li> Reconstitutethe重组人白介素受体α亚基（IL28RA），并根据制造商的说明，并储存于-80℃的重组人干扰素（IFNL1-3）的His标记的配体。 Synthetize阻断肽和如先前所述4中使用。使用PBS中制备不同浓度的配体的秒和在测定中使用的肽。 </li></ol></li><li>为了制备第一抗体，稀释在PBS中的0.1％BSA的6倍他小鼠单克隆抗体以1:1000稀释。为了制备第二抗体，稀释辣根过氧化物酶（HRP）的PBS缀合的山羊抗小鼠IgG（H + L）含有1 0.1％BSA的1:10,000稀释。 </li><li>通过根据制造商的说明混合试剂A和B制备TMB溶液。 </li><li>通过在RT加入在蒸馏水和商店5 N硫酸（H 2 SO 4）准备终止溶液。 </li></ol> 2.酶联免疫测定（ELISA） 注:直接配体-受体相互作用的ELISA（直接LRA， 图1）可以被用于测量受体-配体的解离常数（K D），作为受体-配体结合亲和力的量度。竞争配体-受体相互作用的ELISA（竞争LRA， 图2）的所有流入的肽（和其他阻断化合物），其作用与配体和受体之间的相互作用来干扰的筛选。先前公布5的基本协议进一步优化。 注意:在这两种ELISA方法使用多道移液器用于添加解决方案，以在每一个步骤96孔板的孔中。在溶液澄清或洗涤步骤，直接扔出去的解决方案整合到水槽。 <ol><li>直接配体 - 受体相互作用测定（直接LRA） 注意:对于该工作流的示图（ 见图1）。 <ol><li>涂层板的重组受体<ol><li>稀释在碳酸盐缓冲液的重组受体至100毫微克/微升的最终浓度。通过使用多道移液器以及移液100微升到每个具有固定的受体浓度（100毫微克/微升）96孔微量滴定板的涂层的孔中。排除板外墙面，以免以及边缘神器。覆盖有盖板，孵育的板在4°CO / N。 </li></ol></li><li>阻塞和配体的加入<ol><li>第二天，通过倾斜靠在水槽板除去涂层溶液和3次洗涤液（PBS + 0.05％体积/体积的吐温20）洗涤该板。 </li><li>用200微升的5％BSA溶液到每个用多道移液器井框在涂板的游离受体结合位点和孵育所述板在室温2小时。 </li><li>弃去封闭溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li><li>制备重组His-标记的配体在不同浓度（ 例如，8微克/毫升，4微克/毫升，2微克/毫升，1微克/毫升，0.5微克/毫升，0.25微克/毫升，0.125微克/毫升，0.063微克/毫升，0.031微克/毫升，0.0微克/毫升）的PBS。仅添加PBS的空白孔。 </li><li>加入100微升每配体浓度向孔中一式两份，并在室温孵育2小时的板允许受体 - 配体相互作用。 </li></ol></li><li>孵化与抗体<ol><li>与配体孵育后，3次洗涤平板用洗涤溶液。 </li><li>吸取100μl的初级抗His小鼠单克隆抗体溶液（1:1,000）的每个孔中。 </li><li>孵育在室温下2小时的板;温育后，弃去抗体溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li><li>添加100微升HRP偶联的山羊抗小鼠IgG二抗溶液（1:10,000）到每个孔中。将培养板在室温下45分钟。 </li><li>弃去抗体溶液（见步骤2.1.2.1）和3次洗涤液洗涤该板。 </li></ol></li><li>基材与发展加成<ol><li>使TMB底物溶液到室温，并在1制备TMB底物溶液A和B:1的比例。添加新鲜制备的衬底以每孔100μl，并保持在室温的板15-30分钟。经过充分的山坳<html>或开发加入50微升一站式解决方案。 </li></ol></li><li>读板和数据分析 注意:所述的协议是基于所测量的信号从特异性结合升起的假定。它可能是估算非特异性结合到该信号的贡献必要的，但是这是出于这个协议的范围。 <ol><li>直接读取450nm处的吸光度（光密度，OD值）。 </li><li>减去从测量OD值的背景信号和正常化它们。变换的所有值的配位体浓度为对数标度的（基座10，日志10）。 </li><li>积归一化和背景校正OD值（Y轴，对应于被占领受体结合位点的分数）抗配体浓度的对数（X轴，对数10的范围）。 </li><li>为了估计在K D值，拟合数据到Hill方程的形式如下: tp_upload / 53575 / 53575eq1.jpg"/&gt; 注意:这里Y代表占据受体结合位点和Y 最大最大结合的部分; [L]表示游离配位体和希尔系数的浓度。如果只有一个用于配体结合位点，希尔系数为n = 1，对于具有多于一个的配位体结合位点的系统中，结合展品正协同如果n&gt; 1，负协同如果n &lt;1和没有协同如果n = 1，微观解离常数被称为并对应于半数最大有效浓度EC 50 6。表观解离常数为K D =（K D）ñ。在最简单的，其中n = 1的情况下，解离常数对应于该受体结合位点的一半被占用和KÐ= K D中的配体浓度。这个模型假定质量作用在平衡条件下的结合，以及，只有一小部分加入的配位体与受体结合， 即，[L] &gt;&gt; [RL]。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/53575/53575fig1.jpg" /> 图1. 直接配体-受体相互作用测定（直接LRA）。直接LRA一步一步的协议。 <a href="https://www.jove.com/files/ftp_upload/53575/53575fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>竞争配体 - 受体相互作用测定（竞争LRA） 注意:对于该工作流的示意图参见图2中的竞争LRA程序遵循作为直接LRA除了在配体的重要变化相同的步骤（涂布板，抗体孵育，板发展）和肽加成步骤。合适的阴性对照是用于该测定是必不可少的。在以前的筛选研究&lt;SUP&gt;如图4所示，加扰的封闭肽没有表现出拮抗作用。 <ol><li>阻塞 - 配体和阻止肽加成<ol><li>第二天，除去涂层溶液和洗涤所述板（见2.1.2.1）。 </li><li>通过加入200微升的5％BSA溶液，以每孔阻挡涂板，孵育该板在RT 2小时。 </li><li>在固定的浓度（2×20毫微克/毫升）的PBS制备重组His-标记的配体（IFNL1-3）。 </li><li>制备具有不同浓度范围从10nM至100μM的在PBS中，以保证一个剂量-反应曲线的阻塞肽（参照表3）。 注:此启用阻断肽的IC 50值的连续测定。在对照孔，只加固定的配位体浓度没有肽获得最大（100％）的结合。在一片空白，只添加PBS没有配体或肽。 </li><li>加的配位体的50微升（IFNL1-3）和50微升各PE的ptide集中在重复的井。 </li><li>将培养板在室温2小时。 </li></ol></li><li>读板和数据分析 注意:所述的协议是基于所测量的信号从特异性结合升起的假定。它可能是估算非特异性结合到该信号的贡献必要的，但是这是出于这个协议的范围。 <ol><li>直接读取450nm处的吸光度（光密度，OD值）。 </li><li>减去从测量OD值的背景信号和正常化它们。变换的所有值的肽浓度对数标度的（基座10，日志10）。 </li><li>积归一化和背景校正OD值（Y轴，对应于被占领受体结合位点的分数）抗配体浓度的对数（X轴，对数10的范围）。 </li><li>来估计IC 50值，与数据拟合到以下公式: <img alt="公式（2）" src="/files/ftp_upload/53575/53575eq2.jpg" /> 注:此处[P]为肽浓度和Hill斜率。 Hill斜率描述了剂量 - 反应曲线的陡度。将IC 50对应于被观察到的配体和受体之间的结合的50％抑制的抑制剂浓度。 </li></ol></li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53575/53575fig2.jpg" /> 图2.竞争配体-受体相互作用测定（竞争LRA）。竞争LRA一步一步的协议。 <a href="https://www.jove.com/files/ftp_upload/53575/53575fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<ol>
	<li>Schneider, P., Willen, L., Smulski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+techniques+to+study+ligand-receptor+interactions+and+receptor+activation+by+TNF+superfamily+members.">Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.</a> Methods in enzymology. 545, 103-125 (2014).</li><li>Rossi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+analysis+of+anti-citrullinated+protein/peptide+antibody+affinity.">Biosensor analysis of anti-citrullinated protein/peptide antibody affinity.</a> Analytical biochemistry. 465, 96-101 (2014).</li><li>van der Merwe, P. A., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-adhesion+molecule+interactions+using+surface+plasmon+resonance.">Analysis of cell-adhesion molecule interactions using surface plasmon resonance.</a> Curr Opin Immunol. 8, 257-261 (1996).</li><li>Egli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-28B+is+a+key+regulator+of+B-+and+T-cell+vaccine+responses+against+influenza.">IL-28B is a key regulator of B- and T-cell vaccine responses against influenza.</a> PLoS Pathog. 10, e1004556 (2014).</li><li>Rosenbluh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positively+charged+peptides+can+interact+with+each+other,+as+revealed+by+solid+phase+binding+assays.">Positively charged peptides can interact with each other, as revealed by solid phase binding assays.</a> Analytical biochemistry. 352, 157-168 (2006).</li><li>Goutelle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation:+a+review+of+its+capabilities+in+pharmacological+modelling.">The Hill equation: a review of its capabilities in pharmacological modelling.</a> Fundamental &amp; clinical pharmacology. 22, 633-648 (2008).</li><li>Levin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+derived+from+HIV-1+integrase+that+bind+Rev+stimulate+viral+genome+integration.">Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.</a> PLoS One. 4, e4155 (2009).</li><li>Egli, A., Santer, M. D., O'Shea, D., Tyrrell, D. L., Houghton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+the+interferon-lambda+family+on+the+innate+and+adaptive+immune+response+to+viral+infections.">The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections.</a> Emerging infectious diseases. , e51 (2014).</li><li>Gad, H. H., Hamming, O. J., Hartmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+human+interferon+lambda+and+what+it+has+taught+us.">The structure of human interferon lambda and what it has taught us.</a> J Interferon Cytokine Res. 30, 565-571 (2010).</li><li>Folch, B., Rooman, M., Dehouck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostability+of+salt+bridges+versus+hydrophobic+interactions+in+proteins+probed+by+statistical+potentials.">Thermostability of salt bridges versus hydrophobic interactions in proteins probed by statistical potentials.</a> Journal of chemical information and modeling. 48, 119-127 (2008).</li><li>Yuzlenko, O., Lazaridis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+ionizable+amino+acid+side+chains+at+a+lipid+bilayer-water+interface.">Interactions between ionizable amino acid side chains at a lipid bilayer-water interface.</a> The journal of physical chemistry. B. 115, 13674-13684 (2011).</li><li>Tissot, A. C., Vuilleumier, S., Fersht, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+two+buried+salt+bridges+in+the+stability+and+folding+pathway+of+barnase.">Importance of two buried salt bridges in the stability and folding pathway of barnase.</a> Biochemistry. 35, 6786-6794 (1996).</li></ol>

A.E. was supported by a research grants from the "SNSF Ambizione Score" program (PZ00P3_154709), "Forschungsfond, F&#246;rderung strategischer Projekte" University of Basel, Stiftungsinfektionskrankheiten Basel, and Bangeter Rhyner Stiftung. J.L. acknowledges support by an iPhD fellowship of the SystemsX.ch initiative in systems biology program (9th call).

ELISA是许多实验室标准和行之有效的方法。我们进一步修改和完善了先前公布的方法5,7。所表现出的一步一步的协议说明了如何能够以简单的方式被用于确定配体-受体相互作用的K个D取值。此外，阻挡肽，与配体 - 受体相互作用干扰的IC 50可被确定。 主要优点是快速安装，易于制备的试剂和熟悉的操作，因为大多数研究人员以前使用的ELISA协议。直接LRA协议是...

信号通路的全面了解需要有关配体 - 受体相互作用的详细知识。评估其特异性受体的特定配体的相互作用的大多数方法是昂贵的，耗时的，劳动密集型的，并且需要特定的设备和专门知识1。 本文介绍了两个快速和可靠的逐点的协议来调查的基础上的一种酶的配体 - 受体相互作用酶联免疫吸附测定（ELISA）:直接配体 - 受体相互作用测定（LRA）和竞争LRA。 ELISA是一个高度敏感的，具体的和随时可用的技术，几乎在每一个实验室常规使用。 ELISA可以执行并适于各种方式。所提出的协议对不同的λ干扰素（INFLs）和它们的受体之间的相互作用的调查进行了优化。 直接LRA允许一个quantificati关于配体 - 受体的相对于配体浓度结合和由此产生的结合曲线。使用适当的型号为配体-受体相互作用，可以将数据进一步分析以估计的解离常数（K D）。 在所提出的协议，通常使用希尔方程被施加到该配体 - 受体结合建模。尽管其它方法，例如表面等离子共振技术2,3-允许两个蛋白之间的结合亲和力的测定，这种技术常常是劳动密集型的，昂贵的，并且需要特殊的实验室设备。 竞争LRA能够抑制肽的筛选:在配体 - 受体结合相对于肽浓度定量。这产生描述的肽的抑制作用的剂量 - 反应曲线。数据可以被进一步分析以估计半数最大抑制浓度（IC 50 </子&gt;）阻断肽。 两者的ELISA协议是易于使用，并且可以适合于广泛的研究问题。任何形式的重组蛋白可以用于可靠地并快速确定相互作用的部件。此外，竞争LRA可用于通过使用阻断肽，其目的是为了模仿或配体或受体，以确定配体和受体的关键相互作用位点。如果封闭肽显示有效和特异性抑制，肽占据配体的关键相互作用位点（如肽模拟物的受体）或配体（如肽模拟物的配位体）。 第一个协议描述不同INFLs的在K D值的决心和其受体α亚基， 即使用直接LRA白细胞介素28受体（IL28RA）。接着，第二协议显示如何确定一个20个氨基酸长的肽的能力抑制INFL-IL28RA相互作用。肽被设计成在其受体结合位点与IFNLs竞争，从而使相互作用的分子的理解。此外，该肽可以用于阻止在体外实验IL28RA以确定对下游信号效应4的影响。

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Nunc-Immunoplate (F96 Maxi sorp)</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:119px;">442404</td>
			<td style="width:359px;">ELISA plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium carbonate (Na2CO3)</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">497-19-8</td>
			<td>For ELISA plate coating buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Sodium hydrogen carbomnate(NaHCO3)</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">144-55-8</td>
			<td>For ELISA plate coating buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">A7030-100G</td>
			<td>5% BSA in PBS for Blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28R&#945;/IFN&#955;R1</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">5260-MR</td>
			<td>Recombinant human interlukin-28 Receptor alpha</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-29/IFN&#955;1</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">1598-IL/CF</td>
			<td>Recombinant human interlukin-29/Carrier free/C-terminal 10-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28A/IFN&#955;2</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">1587-IL/CF</td>
			<td>Recombinant human interlukin-28A/Carrier free/C-terminal 6-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28B/IFN&#955;3</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">5259-IL/CF</td>
			<td>Recombinant human interlukin-28B/Carrier free/C-terminal 6-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">6X His Monoclonal antibody (Mouse)</td>
			<td style="width:164px;">Clontech</td>
			<td style="width:119px;">631212</td>
			<td>Primary antiboy to capture His tagged Ligands</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Goat anti-Mouse igG (H+L)</td>
			<td style="width:164px;">Jackson Immuno Research</td>
			<td style="width:119px;">115-035-166</td>
			<td>Horseradish Peroxidase conjucated secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">BDoptEIA TMB reagent set</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:119px;">555214</td>
			<td>ELISA - TMB substrate solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sulfuric acid (H2SO4)</td>
			<td style="width:164px;">Fulka</td>
			<td style="width:119px;">84720</td>
			<td>5N H2SO4 (Enzyme reaction stop solution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Synergy/H1 - Microplate reader</td>
			<td style="width:164px;">BioTeK</td>
			<td style="width:119px;"></td>
			<td>ELISA plate reader</td>
		</tr>
	</tbody>
</table>

an elisa based binding and competition method to rapidly determine ligand-receptor interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

使用直接LRA确定INFL1-3及其受体α亚基IL28RA之间的解离常数。结果示于图3:所占据的结合位点的小数值靠着相应的IFN浓度的对数作图。数据的Scatchard作图示于右下角。的结果表明，直接LRA产生一个结合曲线，其可以被进一步分析，以估计在K D值。在K D值，通过拟合数据至希尔方程（方程1）测定。 IFNL...

Watch this Scientific Journal Video about An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions at JoVE.com

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

的ELISA基结合和竞争法快速测定配体 - 受体相互作用

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

an-elisa-based-binding-competition-method-to-rapidly-determine-ligand

Research

JoVE Journal

Chemistry

18.9K 次观看.  University of Basel. The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions. 

视频: An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

科研

化学

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

质谱途径研究蛋白质结构和相互作用的冻干粉

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

A&#39;即插即用&#39;的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements are simple high-throughput characterization experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glassy states for a variety of glassy materials. This technique allows for these parameters to be measured in a single experiment, while other methods must combine a variety of different techniques to investigate all of these properties. Measurements of dynamics close to Tg are particularly challenging. The advantage of cooling rate dependent Tg measurements over other methods which directly probe bulk and surface relaxation dynamics is that they are relatively quick and simple experiments, which do not utilize fluorophores or other complicated experimental techniques. Furthermore, this technique probes the average dynamics of technologically relevant thin films in temperature and relaxation time (&#964;&#945;) regimes relevant to the glass transition (&#964;&#945; &#62; 100 sec). The limitation to using ellipsometry for cooling rate dependent Tg experiments is that it cannot probe relaxation times relevant to measurements of viscosity (&#964;&#945; &#60;&#60; 1 sec). Other cooling rate dependent Tg measurement techniques, however, can extend the CR-Tg method to faster relaxation times. Furthermore, this technique can be used for any glassy system so long as the integrity of the film remains throughout the experiment.

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

冷却率相关的椭偏仪测量确定薄膜玻膜的动力学

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

对于未螯合钆基于核酸适配体传感器（III）

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

直接溶液还原法快速合成金、钯、铂气凝胶的研究

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...