Name: an electrochemiluminescence-based assay for mecp2 protein variants
Uploaded: 2020-05-22T12:30:00
Description: Watch this Scientific Journal Video about An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants at JoVE.com

我们非常感谢布里吉特·斯特姆博士对ECLIA文书的支持。

澳大利亚韦斯特米德儿童医院人体研究伦理委员会批准了为研究目的进行皮肤活检采购。动物实验的同意得到了奥地利联邦科学、研究和经济部的同意，这些实验是根据当地动物福利条例（GZ：66.009/0218-II/3b/2015）进行的。
注：图1中描述了ECLIA系统的原理。
1. 抗体选择
<ol><li>评估各种 MeCP2 抗体的信号噪声比，并在主小鼠单克隆 MeCP2...

<ol>
	<li>Debad, J. D., Glezer, E. N., Wohlstadter, J., Sigal, G. B., Leland, J. K., Bard, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+biological+applications+of+ECL.">Clinical and biological applications of ECL.</a> Electrogenerated Chemiluminescence. , 359-383 (2004).</li><li>Yuzaburo, N., Michinori, U., Osamu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Electrochemiluminescence+Immunoassay+Using+the+Ruthenium+Chelate-Labeled+Antibody+Bound+on+the+Magnetic+Micro+Beads.">Highly Sensitive Electrochemiluminescence Immunoassay Using the Ruthenium Chelate-Labeled Antibody Bound on the Magnetic Micro Beads.</a> Analytical Sciences. 15 (11), 1087-1093 (1999).</li><li>Liu, W., Hu, Y., Yang, Y., Hu, T., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+immunoassays+for+quantification+of+hepatitis+B+surface+antigen+in+Chinese+patients+with+concomitant+hepatitis+B+surface+antigen+and+hepatitis+B+surface+antibodies.">Comparison of two immunoassays for quantification of hepatitis B surface antigen in Chinese patients with concomitant hepatitis B surface antigen and hepatitis B surface antibodies.</a> Archives of Virology. 160 (1), 191-198 (2015).</li><li>Shah, R. R., Bird, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MeCP2+mutations:+progress+towards+understanding+and+treating+Rett+syndrome.">MeCP2 mutations: progress towards understanding and treating Rett syndrome.</a> Genome Medicine. 9 (1), 17 (2017).</li><li>Chahrour, M., 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=MeCP2,+a+key+contributor+to+neurological+disease,+activates+and+represses+transcription.">MeCP2, a key contributor to neurological disease, activates and represses transcription.</a> Science. 320 (5880), 1224-1229 (2008).</li><li>Lyst, M. J., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome:+a+complex+disorder+with+simple+roots.">Rett syndrome: a complex disorder with simple roots.</a> Nature Reviews Genetics. 16 (5), 261-275 (2015).</li><li>Katz, D. M., 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=Rett+Syndrome:+Crossing+the+Threshold+to+Clinical+Translation.">Rett Syndrome: Crossing the Threshold to Clinical Translation.</a> Trends in Neurosciences. 39 (2), 100-113 (2016).</li><li>Steinkellner, 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=An+electrochemiluminescence+based+assay+for+quantitative+detection+of+endogenous+and+exogenously+applied+MeCP2+protein+variants.">An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.</a> Scientific Reports. 9 (1), 7929 (2019).</li><li>Laccone, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Synthetic MeCP2 sequence for protein substitution therapy. , (2007).</li><li>Koutsokeras, A., Kabouridis, 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=Secretion+and+uptake+of+TAT-fusion+proteins+produced+by+engineered+mammalian+cells.">Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790 (2), 147-153 (2009).</li><li>Suzuki, K., Bose, P., Leong-Quong, R. Y., Fujita, D. J., Riabowol, K. R. E. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REAP:+A+two+minute+cell+fractionation+method.">REAP: A two minute cell fractionation method.</a> BMC Research Notes. 3, 294 (2010).</li><li>Xia, H., Mao, Q., Davidson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV+Tat+protein+transduction+domain+improves+the+biodistribution+of+beta-glucuronidase+expressed+from+recombinant+viral+vectors.">The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors.</a> Nature Biotechnology. 19 (7), 640-644 (2001).</li><li>Schwarze, S. R., Ho, A., Vocero-Akbani, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+transduction:+delivery+of+a+biologically+active+protein+into+the+mouse.">In vivo protein transduction: delivery of a biologically active protein into the mouse.</a> Science. 285 (5433), 1569-1572 (1999).</li><li>Nagahara, 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=Transduction+of+full-length+TAT+fusion+proteins+into+mammalian+cells:+TAT-p27Kip1+induces+cell+migration.">Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration.</a> Nature Medicine. 4 (12), 1449-1452 (1998).</li><li>Trazzi, 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=CDKL5+protein+substitution+therapy+rescues+neurological+phenotypes+of+a+mouse+model+of+CDKL5+disorder.">CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder.</a> Human Molecular Genetics. 27 (9), 1572-1592 (2018).</li><li>Van Esch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MECP2+Duplication+Syndrome.">MECP2 Duplication Syndrome.</a> Molecular Syndromology. 2 (3-5), 128-136 (2012).</li><li>Collins, A. 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=Mild+overexpression+of+MeCP2+causes+a+progressive+neurological+disorder+in+mice.">Mild overexpression of MeCP2 causes a progressive neurological disorder in mice.</a> Human Molecular Genetics. 13 (21), 2679-2689 (2004).</li><li>Luikenhuis, S., Giacometti, E., Beard, C. F., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+MeCP2+in+postmitotic+neurons+rescues+Rett+syndrome+in+mice.">Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6033-6038 (2004).</li></ol>

为了测量内源性MeCP2、重组MeCP2和TAT-MeCP2水平，开发了96孔板ECLIA。已经表明，MeCP2蛋白功能的丧失导致RTT综合征6，目前治疗仅限于症状管理和物理治疗。一个有前途的治疗途径是所谓的蛋白质替代疗法，其中MeCP2水平可以打分到他们所需的浓度12，13，14，15。12,13,14,15过去二十年来，TA...

电化学发光免疫测定（ECLIA）基于一个过程，该工艺利用标签，设计在电化学刺激时发出发光。它是一种广泛适用的生物分析物定量检测技术，用于基础工业和学术研究、食品工业以及临床诊断1。通常，使用带有碳墨水电极的一次性 96 孔板。这些电极充当免疫测定的固相载体。二次抗体与电化学发光标签结合，当电应用于系统时，触发化学标签的光发射。超低噪声电荷耦合装置（CCD）记录光强度与与捕获抗体结合的抗原成正比，从而对样品2的目标解解物进行定量。与酶相关的免疫吸附剂测定（ELISA）相比，ECLIA被认为是有利的，因为它提供了更高的灵敏度和可重复性，以及更好的自动化和一致性3。
在这里，我们分析了人类和鼠源样品中的甲基-CpG结合蛋白2（MeCP2）水平，以及使用新开发的ECLIA系统对重组蛋白的不同变异。MecP2是一种与X相关的核酸结合蛋白，已知与甲基化DNA序列相互作用。这种蛋白质已经牵连到基因表达的调节44，5。5编码这种蛋白质的基因功能丧失突变是导致Rett综合征（RTT）的罪魁祸首，一种严重的神经发育障碍6。另一种MeCP2相关疾病，MECP2重复综合征，也导致神经症状，可以重叠的RTT7。值得注意的是，女性大多受RTT影响，而男性则大多受MECP2重复综合征66、77的影响。
这些疾病分别与中枢神经系统（CNS）中MeCP2水平不足或过量有关。因此，RTT的治疗方案，涉及增加MECP2水平在CNS将需要避免与过量的MeCP27相关的有害影响。由于这一事实，ECLIA系统对MeCP2蛋白水平进行高度敏感和准确的定量，对于推进RTT以及MECP2重复综合征研究至关重要。从人体细胞系和小鼠组织样本中精确测量内源性和外源性MeCP2水平，以及由人类MeCP2等型B（也称为异体e1）组成的重组蛋白，以及具有跨越血脑屏障88、99的最小N-终端HIV-TAT转导域（TAT-MeCP2）。

<table>
	<tbody>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>500-0006</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Detection AB SULFO-TAG labeled anti-rabbit</td>
			<td>Meso Scale Diagnostics</td>
			<td>R32AB-1</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Discovery workbench 4.0</td>
			<td>Meso Scale Discovery</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>DMEM (1X)</td>
			<td>gibco by Life Technologies</td>
			<td>41966-029</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td>Sample preperation, Washing solution, Coating solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G2025</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>Gold Read Buffer T (1x) with surfactant</td>
			<td>Meso Scale Diagnostics</td>
			<td>R92TG</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>KIMBLE Dounce tissue grinder set</td>
			<td>Sigma-Aldrich</td>
			<td>D8938</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Laboratory Shaker, rocking motion (low speed)</td>
			<td>GFL</td>
			<td>3014</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl*6H20)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>MeCP2 (Human) Recombinant Protein (P01)</td>
			<td>Abnova Corporation</td>
			<td>H00004204-P01</td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Microseal B seal</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>MSB1001</td>
			<td>for plate sealing</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin</td>
			<td>Sigma-Aldrich</td>
			<td>M6818-100UL; RRID:AB_262075</td>
			<td>Antibody, Coating solution</td>
		</tr>
		<tr>
			<td>MSD Blocker A</td>
			<td>Meso Scale Diagnostics</td>
			<td>R93BA-4</td>
			<td>Blocker</td>
		</tr>
		<tr>
			<td>MSD SECTOR Imager 2400</td>
			<td>Meso Scale Diagnostics</td>
			<td>I30AA-0</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Multi-Array 96-well Plate</td>
			<td>Meso Scale Diagnostics</td>
			<td>L15XB-3/L11BX-3</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>gibco by Life Technologies</td>
			<td>15140122</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Polyclonal Anti-MeCP2, produced in rabbit</td>
			<td>Eurogentec S.A.</td>
			<td>custom-designed</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Merck KGaA</td>
			<td>1049361000</td>
			<td>Hypotonic lysis reagent</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (1B11)</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1404063; RRID:AB_10737296</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (4B6)</td>
			<td>Sigma-Aldrich</td>
			<td>WH0004204M1; RRID:AB_1842411</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (Mec-168)</td>
			<td>Sigma-Aldrich</td>
			<td>M6818; RRID:AB_262075</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (Men-8)</td>
			<td>Sigma-Aldrich</td>
			<td>M7443; RRID:AB_477235</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Rabbit, anti-MeCP2 (D4F3)</td>
			<td>Cell Signaling Technology</td>
			<td>3456S; RRID:AB_2143849</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail (100X)</td>
			<td>Sigma-Aldrich</td>
			<td>8340</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>Secondary AB, Rabbit, anti-MeCP2</td>
			<td>Eurogentec S.A.</td>
			<td>custom</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Secondary AB, Rabbit, anti-MeCP2</td>
			<td>Merck</td>
			<td>07-013</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>SULFO-TAG Labeled Anti-Rabbit Antibody (goat)</td>
			<td>Meso Scale Diagnostics</td>
			<td>W0015528S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>TAT-MeCP2 fusion protein</td>
			<td>in-house production</td>
			<td></td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25% (1X)</td>
			<td>gibco by Life Technologies</td>
			<td>25200-056</td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td>Washing solution</td>
		</tr>
	</tbody>
</table>

an electrochemiluminescence-based assay for mecp2 protein variants

ECLIA 是一种多功能方法，能够以 96 孔格式量化内源和重组蛋白量。为了证明ECLIA的效率，该测定用于分析小鼠脑组织中MeCP2的内在水平和TAT-MeCP2在人类皮肤纤维细胞中的接受。MeCP2-ECLIA 可生成高精度和可重复的测量，具有较低的内部和内部测定误差。总之，我们开发了一种定量方法，用于评估MeCP2蛋白变异，可用于高通量屏幕。

图1描述了ECLIA系统的原理。图 2显示了两个 MeCP2 变体的标准曲线。在广泛的浓度（1~1，800纳克/mL）上可以精确定量。在图3中，分析了从小鼠大脑和HDF中提取的MeCP2水平裂灰。图3A对来自异质细胞、野生型和敲空小鼠的脑核结扎液中的MeCP2表达进行了对比，而图3B中，使用ECLIA的MECP2缺乏人...

Watch this Scientific Journal Video about An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants at JoVE.com

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

电化学发光免疫测定（ECLIA）是一种定量检测内源和外源应用的MeCP2蛋白变异的新方法，在广泛的工作范围内产生高定量、准确和可重复的测量，检测内误差低。此处介绍了 MeCP2-ECLIA 的 96 井格式的协议。

MeCP2蛋白质变异的电化学发光测定

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

ECLIA 是一种简单而有效的方法来量化 MeCP2 的细胞水平。与其他定量方法（如西方印迹和 ELISA）相比，ECLIA 速度快、更灵敏、更易于处理。要准备洗涤溶液，即 PBS 中的 0.5%Tween 20，请将 500 微升 Tween 20 添加到一升 PBS 中，并大力混合。准备 3%阻滞器 A 和 PBS 的阻塞溶液，轻轻搅拌。通过过滤对堵塞溶液进行消毒。要准备测定稀释剂溶液，1%阻滞器 A 和 PBS，请向 10 毫升 PBS 添加 5 毫升的阻滞溶液。接下来在冰上解冻单克隆小鼠抗 MeCP2 抗体。将抗体的0.67微升与4毫升PBS混合，然后涡旋溶液。为了溶液涂覆96井的高粘合板，使用多通道管道将25微升涂层溶液分入每井的底角。将 96 井板轻轻涂抹在每侧，以确保涂层溶液覆盖每一个井的底部。用胶箔密封板，在摄氏四度下孵育过夜。从冰箱中取出 96 井板并取出铝箔。将抗体涂层溶液轻拂到废容器中，将其去除。然后点按纸巾上的盘子，取出任何剩余的溶液。接下来，向每井添加 125 微升的阻塞解决方案。然后用粘合箔再次密封板。将板放在轨道微孔板摇床上，设定为 800 RPM，并在室温下孵育 90 分钟。在冰上解冻一小瓶 MeCP2 或 Tat-MeCP2 蛋白质库存溶液、小鼠大脑解酶和 HDF 解酶。稀释清洁管中的蛋白质储存溶液，如手稿表2所述。接下来稀释落酸样品。对于小鼠大脑解液，每25微升的解解缓冲液使用1至20微克。对于 HDF 解酸，每 25 微升解液缓冲液中加入 0.25 至 1 微克。96 孔板孵育 90 分钟后，将堵塞溶液从孔中去除，将其轻拂到废物容器中。然后点按纸巾上的盘子，取出任何剩余的溶液。然后用150微升的洗涤液洗三次，加入洗涤液并立即取出。在96孔板的井中加入25微升的标准和样品。密封板，并在室温下再孵育四小时，并持续 800 RPM 的震动。在冰上解冻未标记的检测抗体，多克隆兔抗MCP2。使用测定稀释剂溶液，以1比6000的比例稀释抗体。当 96 井板在摇床上孵育完成后，通过将板轻拂到废容器中去除标准和样品。然后点按纸巾上的盘子，取出任何剩余的溶液。加入洗涤液并立即取出，用150微升的洗涤溶液洗三次。使用多通道管道机将 25 微升无标签检测抗体溶液添加到每一井。密封板并在室温下孵育一小时，在 800 RPM 下持续摇晃。从冰箱获得特定的二级抗体，并放置一块冰。在测定稀释剂溶液中稀释辅助体，轻轻混合。要从 96 井板中去除无标签检测抗体，请轻拂到废容器中，然后用纸巾轻点板。洗涤板三次后，用多通道管道机将25微升的二次抗体添加到每井中。密封板并在室温下孵育一小时，在 800 RPM 下持续摇晃。去掉释放的二次抗体，如前文所述，轻拂到废物容器中，用纸巾敲击盘子，然后洗三次盘子。在板的每个井中，加入150微升1X Tris基金读取缓冲器与表面活性剂，含有三叶胺作为光生成的核心活性剂。为避免产生气泡，请使用反向移液技术。使用电化学发光检测系统，将 96 孔板放在微孔板上。使用内置的 CCD 摄像机，立即开始捕获数据。记录对应于相对光单位且与光强度成正比的信号计数。MeCP2 标准曲线是在多次测量中从人类 MeCP2 生成的。ECLIA 可以精确量化重组人类 MeCP2 的范围，从每毫升一毫克到每毫升 1800 毫微克。使用ECLIA，MeCP2蛋白水平测量在脑核酸盐从异质和雌性野生型小鼠和一个梅CP2淘汰小鼠。ECLIA是在细胞酸盐上进行的，来自健康对照组，以及用于使用为雷特综合征模型的c.806delG细胞系。ECLIA或免疫荧光在突变细胞系中未检测到 MeCP2 蛋白。ECLIA还用于调查 MeCP2 缺陷细胞线路加班对 Tat-MeCP2 的吸收。在连续三天内测定了ECLIA间和内部测定精度。对于未来的蛋白质替代疗法，MeCP2的输送可以在ECLIA进行蛋白质水平评估后反复优化。

Research

JoVE Journal

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

科研

生物化学

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...