我们非常感谢维克多GJ罗杰斯宝贵的意见。我们感谢所​​有的廖组的成员非常密切的合作工作，并帮助这项研究。这项研究是由美国国立卫生研究院（批准AI076504 JL）的支持。

1。质粒的构建。 <ol><li>通过PCR扩增的基因的开放阅读框架，PCR产物克隆到pCRII-TOPO载体中。 </li><li>通过测序确认产品，和克隆的cDNA编码CyPet-（预SUMO1）-YPet，-SUMO1 CyPet，YPet和催化结构域的SENP1到pET28（二）的N-末端六组氨酸标签的向量。 </li></ol> 2。蛋白质表达与纯化<ol><li>转化大肠杆菌菌株BL21（DE3）细胞与pET28（二）编码CyPet-（预SUMO1）-YPet，-SUMO1 CyPet，YPet和催化结构域的SENP1的载体。 </li><li> 3小时的2xYT培养基中生长的转化的细菌在37℃下（在250rpm摇动），达到在600nm处的光密度为0.5。 </li><li>加入100μM的异丙基-β-D-硫代半乳糖苷（IPTG）（最终浓度），以诱导蛋白表达，并在25℃下振摇16小时以200rpm。 </li><li>收获的细菌centrifug振荡器在10,000 rpm下在4℃下5分钟，并再悬浮在一个缓冲区中的20mM Tris-HCl（pH值7.4）中，50 mM氯化钠，和5mM咪唑。 </li><li>在功率设置为25 W使用MiSonics 4000超声波发生器，超声处理10分钟的细胞以5秒的间隔，并通过离心收集它们在35,000 xg离心在4℃下进行30分钟。 </li><li>设置列用500毫升的Ni-NTA珠为1 L培养上清液转移入列。 </li><li>的20mM Tris-HCl（pH值7.4）中，500 mM氯化钠，10mM咪唑两次用缓冲液洗涤树脂。 </li><li>厄鲁特蛋白质用缓冲液的20mM Tris-HCl（pH值7.4）中，500 mM氯化钠，和500mM咪唑的20mM的Tris-HCl（pH值7.4），50 mM氯化钠和1mM二硫苏糖醇（DTT）到一个缓冲区和透析4°C过夜。 </li><li>通过Bradford法确定的纯化的蛋白质的浓度。蛋白质浓度测量的另一种方法，将SDS-PAGE凝胶电泳，然后用考马斯亮蓝染色，然后用成像定量。 </li></ol> 3。定量荧光共振能量转移频谱分析仪<ol><li>检测的总体战略是基于FRET信号（ 图1）。 FRET对，CyPet和YPet的，被标记的N-和C-末端，分别预SUMO1。 SENP1切割融合蛋白CyPet-（预SUMO1）YPet SUMO1的C-末端的Gly-甘氨酸站点，因此，发行与YPet SUMO的尾巴。 FRET的信号被破坏，导致增加的排放物CyPet和急剧减少YPet的排放的CyPet激发波长。 </li><li>当激发光的波长414nm处，的总荧光发射的CyPet（预-SUMO1）-YPet在530 nm处可以被来自从3来源：的绝对FRET-诱导YPet的排放，CyPet直接排放和YPet直接排放（ 图2）。 </li></ol><img alt="式（1）" src="/files/ftp_upload/4430/4430eq1.jpg" /> 其中FL 四百十四分之五百三十〇 &lt;/ sub&gt;的总荧光发射波长为530 nm时，在414 nm的激发，FL 荧光共振能量转移是绝对的共振信号，：FL CyPet（续）是CyPet直接排放兴奋时，位于414 nm，：FL YPet（续）是在YPet的兴奋时，位于414 nm的直接排放。 （续）下标代表了贡献。 <ol start="3"><li>在414 nm处激发时具有恒定 ​​的比例的α，在530 nm处的直接排放CyPet是正比于它的发射波长在475 nm。CyPet SUMO1制备浓度为50，100，200，500，和750纳米和1mM和排放量在475和530 nm处分别测定激发后在414 nm处测定α（ 图3-1）。 </li><li>在530 nm处的激发下在414 nm处的直接排放YPet成正比其在530 nm处的发射波长在475 nm激发时具有恒定 ​​的比例，β。YPet制备浓度为50，100，200，500，750的届1,000纳米，和发射波长为530 nm，测定时，样品由414和475 nm的波长激发，以确定β（ 图3-2）。 </li><li>由SENP1，当CyPet（预SUMO1）YPet消化，裂解CyPet-SUMO1和SUMO1尾YPet的。当化合物（ 佛罗里达州&#39;530/414）在530 nm处的荧光发射波长在414 nm激发，被减少，但仍然可以被分为三个部分： </li></ol><img alt="公式2" src="/files/ftp_upload/4430/4430eq2.jpg" /> FL&#39; 四百十四分之五百三十零的总消化后兴奋时，位于414 nm的荧光发射波长为530 nm，FL&#39;FRET绝对是剩余的共振信号，FL&#39;CyPet（475/414）是CyPet的排放量为475 nm时，消化后位于414 nm（CyPet排放在这里是由两部分组成：未消化CyPet-（预SUMO1）YPet，消化CyPet-SUMO1）激发和FL YPet（530/475）是在YPet的排放时，在475 nm的激发，这是不变的，是否CyPet-（预SUMO1）YPet消化或不。 <ol start="6"><li>由SENP1消化后，其它FRET发射（FL&#39;FRET）是： </li></ol><img alt="式（3）" src="/files/ftp_upload/4430/4430eq3.jpg" /> 其中 C是总浓度CyPet-（预SUMO1）YPet 和 x是消化CyPet浓度（预SUMO1）YPet。 <ol start="7"><li>通过组合所有的项目中，检测出的荧光发射在530 nm下的414纳米（FL&#39;530/414）的激发是： </li></ol><img alt="式（4）" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4。基于FRET的蛋白酶含量，酶动力学研究<ol><li> CyPet-（预SUMO1）-YPet SENP1的催化结构域的孵育在37℃下在一个缓冲区中的20mM的Tris-HCl（pH值7.4），50 mM氯化钠，0.1％（体积/v）吐温20和1mM DTT的总体积为80ml，并转移到384孔板中。 </li><li>运行，进行了15秒的间隔的5分钟，使用的激发波长为414 nm的荧光多孔板读数器后，通过测量在475和530 nm处的荧光发射。 </li><li>反应速率（v）与在基片（S）的量的变化作为</li></ol><img alt="方程（5）" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li>成倍增加的产物浓度从0为[S] 0（原底物浓度）， 当 t = 0时： </li></ol><img alt="方程（6）" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> 当 t = 0时，原来的速度（V 0）是： </li></ol><img alt="式（7）" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li>的浓固定，SENP1 entration和CyPet-（预SUMO1）-YPet的浓度增加至100倍以上酶的浓度，这是所需的Michaelis-Menten方程式。 </li><li>所有的荧光读数由定量FRET分析方法进行分析，并绘制在V软件GraphPad棱镜适合Michaelis-Menten方程式。比较常见的套装软件，如电子表格程序（如Microsoft Excel）的帮助下，也可以进行非线性回归。 </li></ol> 5。代表性的成果成熟的预SUMO1 SENP1可以在此过程中通过监测在475和530 nm处的荧光信号中的变化确定。结果表明，预SUMO1消化SENP1的速度在基板剂量依赖性的方式（ 图4）。这表明，催化结构域的SENP1预SUMO1成熟表现出极好的活性。最初的反应TION速度计算具有不同的底物浓度（ 表1）通过上述分析。 的 k 猫 / K M比一般用于比较不同的酶的效率，与一个基板或一个特定的酶具有不同的基板，可以得到K M 和 V max通过绘制各种初始速度，对应从Michaelis-Menten方程式的不同浓度的CyPet-（预SUMO1）-YPet（ 图5），K 猫得到作为： <img alt="式（8）" src="/files/ftp_upload/4430/4430eq8.jpg" /> 根据以上的分析，计算出的K M为0.21±0.04μM， 第 k 猫为6.90±0.28秒-1，并且第 k 猫 / K M比为（3.2±0.55）×10 7 M -1的 </sup<html> &gt; -1。 <img alt="图1" src="/files/ftp_upload/4430/4430fig1.jpg" /> 图1。基于FRET的蛋白酶SENP的的前SUMOs成熟的分析图。 <img alt="图2" src="/files/ftp_upload/4430/4430fig2.jpg" /> 图2。的供体，受体和FRET定量分析荧光信号的贡献。夹层的发射光谱从CyPet（预SUMO1）YPet激发下，在414 nm处。：FL CyPet（四百一十四分之五百三十○）， 是CyPet的排放量在530 nm的激发下，414纳米，FL 荧光共振能量转移是在530纳米，414 nm的激发下荧光共振能量转移，诱导YPet排放，FL YPet（四百十四分之五百三十）YPet的排放量在530波长414 nm的激发下。 load/4430/4430fig3.jpg“/&gt; 图3。方向的排放因子α和β的计算。 <img alt="图4" src="/files/ftp_upload/4430/4430fig4.jpg" /> 图4。定量分析CyPet-（预SUMO1）-SENP1的催化结构域的不同比例的消化YPet。在第5分钟反应监测。 <img alt="图5" src="/files/ftp_upload/4430/4430fig5.jpg" /> 图5。米氏图形分析的SENP1 CyPet（预SUMO1）YPet的消化。数据绘制和GraphPad棱镜V和非线性回归分析。 <table border="1"><tbody><tr><td> [S]（μM）的 </td><td> V 0（μM/秒） </td></tr><tr><td> 0.115 </td><td> 0.0023±0.00005 </td></tR&gt; <tr><td> 0.214 </td><td> 0.0028±0.00004 </td></tr><tr><td> 0.407 </td><td> 0.0033±0.00007 </td></tr><tr><td> 0.594 </td><td> 0.0037±0.00013 </td></tr><tr><td> 0.725 </td><td> 0.0043±0.00012 </td></tr><tr><td> 1.471 </td><td> 0.0051±0.00036 </td></tr><tr><td> 1.899 </td><td> 0.0050±0.00031 </td></tr><tr><td> 2.300 </td><td> 0.0050±0.00062 </td></tr></tbody></table> 表1。初始速度确定前SUMO1的成熟SENP1。以各底物浓度，四个样品被用来衡量消化。来自这四个样品的变化的标准偏差。 <table border="1"><tbody><tr><td> K M（μM）的 </td><td> ķ 猫 （1） </td><td> ķ 猫 / K M（M-1•S-1） </td></tr><tr><td> 0.21±0.04 </td><td> 6.90±0.28 </td><td> 3.2±0.55×10 7个 </td></tr></tbody></table> 表2。前SUMO1的成熟SENP1通过定量荧光共振能量转移的动力学参数分析。来自四个样品在每个底物浓度的标准偏差。

<ol>
	<li>Johnson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+By+SUMO.">Protein modification By SUMO.</a> Annual Review of Biochemistry. 73, 355-382 (2004).</li><li>Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+and+ubiquitin+in+the+nucleus:+different+functions,+similar+mechanisms.">SUMO and ubiquitin in the nucleus: different functions, similar mechanisms.</a> Genes &amp; Development. 18, 2046-2059 (2004).</li><li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO:+a+history+of+modification.">SUMO: a history of modification.</a> Molecular Cell. 18, 1-12 (2005).</li><li>M&#252;ller, S., Hoege, C., Pyrowolakis, G., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO,+ubiquitin's+mysterious+cousin.">SUMO, ubiquitin's mysterious cousin.</a> Nature. , 202-210 (2001).</li><li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SP-RING+for+SUMO:+new+functions+bloom+for+a+ubiquitin-like+protein.">SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.</a> Cell. , 5-8 (2001).</li><li>Drag, M., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeSUMOylating+enzymes-SENPs.">DeSUMOylating enzymes-SENPs.</a> IUBMB Life. 60, 734-742 (2008).</li><li>Mukhopadhyay, D., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+reverse:+the+SUMO+proteases.">Modification in reverse: the SUMO proteases.</a> Trends in Biochemical Sciences. 32, 286-295 (2007).</li><li>Reverter, D., Lima, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+SENP2+protease+interactions+with+SUMO+precursors+and+conjugated+substrates.">Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates.</a> Nature Structural &amp; Molecular Biology. 13, 1060-1068 (2006).</li><li>Shen, L., 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=SUMO+protease+SENP1+induces+isomerization+of+the+scissile+peptide+bond.">SUMO protease SENP1 induces isomerization of the scissile peptide bond.</a> Nature Structural &amp; Molecular Biology. 13, 1069-1077 (2006).</li><li>Horton, R., Strachan, E., Vogel, K., Riddle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+for+deubiquitinating+enzymes+based+on+time-resolved+fluorescence+resonance+energy+transfer+between+terbium+and+yellow+fluorescent+protein.">A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.</a> Analytical Biochemistry. 360, 138-143 (2007).</li><li>Mikolajczyk, 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=Small+Ubiquitin-related+Modifier+(SUMO)-specific+Proteases:+PROFILING+THE+SPECIFICITIES+AND+ACTIVITIES+OF+HUMAN+SENPs.">Small Ubiquitin-related Modifier (SUMO)-specific Proteases: PROFILING THE SPECIFICITIES AND ACTIVITIES OF HUMAN SENPs.</a> Journal of Biological Chemistry. 282, 26217-26224 (2007).</li><li>Kolli, N., Mikolajczyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., Salvesen, G., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distrubition+and+paralogue+specificity+of+mammalian+deSUMOylating+enzymes.">Distrubition and paralogue specificity of mammalian deSUMOylating enzymes.</a> Biochem. J. , 335-344 (2010).</li><li>Drag, M., Mikolajczyk, J., Krishnakumar, I. M., Huang, Z., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+profiling+of+human+deSUMOylating+enzymes+(SENPs)+with+synthetic+substrates+suggests+an+unexpected+specificity+of+two+newly+characterized+members+of+the+family.">Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family.</a> Biochemical Journal. 409, 461 (2008).</li><li>Engels, I. H., 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=A+time-resolved+fluorescence+resonance+energy+transfer-based+assay+for+DEN1+peptidase+activity.">A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.</a> Analytical Biochemistry. 390, 85-87 (2009).</li><li>Martin, S., Hattersley, N., Samuel, I., Hay, R., Tatham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-resonance-energy-transfer-based+protease+activity+assay+and+its+use+to+monitor+paralog-specific+small+ubiquitin-like+modifier+processing.">A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.</a> Analytical Biochemistry. 363, 83-90 (2007).</li><li>Nguyen, A. W., Daugherty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nature Biotechnology. 23, 355-360 (2005).</li></ol>

没有利益冲突的声明。

FRET技术用于研究前SUMO1的成熟，由SENP1 9。 CFP-YFP用作FRET对和比例的分析，这是受体捐助排放比，用于表征的动力学性质。然而，有没有考虑供体和受体在传统比例的自身荧光共振分析。比例不直接与衬底消化量。 在这里，我们报告一个高度敏感的基于FRET的的蛋白酶分析研究动力学的前SUMO1的成熟SENP1。对比的比例的方法，从根本上提高一个新的理论方法与共振信号?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PCR II TOPO包</td><td> Invitrogen公司</td><td> K466040 </td></tr><tr><td>的2xYT </td><td>研究产品Internationa.l公司</td><td> X15600 </td></tr><tr><td>镍-NTA琼脂糖</td><td> Thermo Scientific的</td><td> 88222 </td></tr><tr><td>考加号（布拉德福德）含量摄政</td><td> Thermo Scientific的</td><td> 23238 </td></tr><tr><td> 384孔板（玻璃底） </td><td>格雷纳</td><td> 781892 </td></tr><tr><td> FlexStation II 384酶标仪</td><td>分子器件</td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

可逆的翻译后修饰的蛋白质与泛素或泛蛋白 - 样的蛋白质（Ubls）被广泛地用于动态调节蛋白的活性，并具有在许多生物过程中的各种不同的角色。例如，SUMO共价键修改大量蛋白质的重要​​角色，在许多细胞过程，包括细胞周期调控，细胞的生存和死亡，DNA损伤反应和应激反应1-5。 SENP，SUMO特异性蛋白酶，成熟的SUMO前体肽链内切酶的功能，或作为isopeptidase删除SUMO从靶蛋白SUMO化循环刷新1,3,6,7。 或特异性的酶的催化效率最好是其特征在于由比率的动力学常数，K 猫 / K M。在几项研究中，的动力学参数的SUMO-SENP对已确定的各种方法，包括基于聚丙烯酰胺凝胶Western-blot检测，RADIOActive的标记底物，荧光化合物或蛋白质标记底物8-13。然而，聚丙烯酰胺凝胶为基础的技术，它使用“本土”的蛋白质，但费力，技术要求高，不容易借给自己详细的定量分析。所得到的K 猫 / K M从使用的四肽或蛋白的研究，与ACC（7 -氨基-4-carbamoylmetylcoumarin）或AMC（7 -氨基-4 -甲基香豆素）荧光团要么达两个数量级低于天然底物或不能明确区分和肽链内切酶的活动SENPs。 最近，基于FRET的蛋白酶检测使用，研究去泛素酶（DUBS）或SENPs的FRET对青色荧光蛋白（CFP），黄色荧光蛋白（YFP）9,10,14,15。受体发射的比率被用作供体发射的定量参数FRET蛋白酶交流信号监视器拉了一的决心。然而，这种方法忽略的信号交叉污染的受体和供体受体和供体荧光发射波长，因而是不准确的。 我们开发了一个新的高度基于FRET的蛋白酶敏感和定量分析，确定动力学参数的SUMO1成熟的SENP1。一种工程FRET对CyPet的和YPet显着提高的FRET效率和荧光量子产率，被用来生成的CyPet（预SUMO1）-YPet基板16。我们差异化和量化的绝对贡献的供体和受体和FRET在接受器和发射波长，荧光信号。得到的k 猫 / K M的值（3.2±0.55）×10 7 M -1 -1 SENP1朝向预SUMO1，这是与一般的酶动力学参数。因此，这种方法是有效的，并可以使用了一个通用的方法来描述以及其他蛋白酶。

Watch this Scientific Journal Video about Quantitative FRET FÃ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

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

涉及的FRET（福斯特共振能量转移）信号的定量分析酶动力学研究的一种新方法。<em&gt; K<sub&gt; M</sub</em&gt;和<em&gt; K<sub&gt;猫</sub</em&gt; SENP1（SUMO / Sentrin特定的蛋白酶1）预SUMO1（小泛素样修饰）的催化结构域的水解获得。这个定量的基于FRET的蛋白酶动力学研究的一般原则可以应用到其他的蛋白酶。

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 ...

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

Unit 1

Work and Kinetic Energy

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18.9K 次观看.  University of California, Riverside. 涉及的FRET（福斯特共振能量转移）信号的定量分析酶动力学研究的一种新方法。 K M

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

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

改进的可视化和定量分析，药物使用Micropatterned细胞的影响

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

科研

生物工程

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

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

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

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 ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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. ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...