Name: ultra-high-speed western blot using immunoreaction enhancing technology
Uploaded: 2020-09-26T13:00:00
Description: Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

这项工作得到了国家卫生研究院内脏研究司、国家心脏、肺和血液研究所的支持。S.H.得到日本公私合作学生出国留学计划的支持，H.N.和K.Y.获得Valor和V药物海外培训奖学金。

1. SDS-PAGE 并传输到 PVDF 膜
<ol><li>执行 SDS-PAGE，根据相对大小分离蛋白质。 注意：任何商业或自制凝胶系统都没问题。请遵守制造商的协议。</li>
	<li>将分离的蛋白质从凝胶转移到PVDF膜上。 注意：常见的转移方法（半干或湿转移）是合适的。请遵守制造商的协议。</li>
</ol>2. 阻塞PVDF膜
<ol><li>在室温下搅拌时，将膜浸泡在适当的阻塞缓冲区中1小时。 注：根据主要抗体和?...

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, S. 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=Old+but+not+Obsolete:+An+Enhanced+High+Speed+Immunoblot.">Old but not Obsolete: An Enhanced High Speed Immunoblot.</a> Journal of Biochemistry. 168 (1), 15-22 (2020).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+Procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., 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+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. 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西方污点是一种广泛使用的分析技术，用于检测40年前7、8年前开发的特定蛋白质。此后，该技术不断发展，随后的创新提高了技术2、9、10、11、12、13的灵敏度、速度和定量。此处介绍的增强型超高速西?...

西方的污点（也称为免疫虫）是一种强大而基本的技术，跨越广泛的科学和临床学科。该技术用于检查蛋白质1的存在、相对丰度、相对分子质量和转化后修饰。结合数字图像分析，该方法可以可靠地分析蛋白质的丰度和蛋白质的修饰2。虽然西方的污点是例行公事，但它是一种耗时和劳动密集型的方法。需要用膜长期培养抗体。在这里，我们描述了对孵化方法的修改，该方法克服了这一限制，而不会牺牲灵敏度。
在膜的孵化过程中，抗体漂浮在溶液中，而抗原则固定在膜上。由于其亲和力高，抗体与抗原结合的速度比抗体从散装溶液扩散到膜的速度要快。这会产生低浓度的"消耗层"（图 1）。较远的抗体可能需要数小时才能通过被动扩散到达膜，而被动扩散是导致该技术长期潜伏的主要因素。此效果称为大众运输限制 （MTL）3。有人提出，重复排空和补充含抗体溶液可能会破坏耗竭层，克服MTL4。在这里，我们设计了一种独特的技术，实现这种循环排水和补充 （CDR） 概念，以减少 MTL 在传统的西方印迹协议中的影响，并显著缩短所需的潜伏期。
为了在短的潜伏时间内保持检测灵敏度，我们采用了提高免疫力增强技术，加速抗原抗体反应，从而提高了信号噪声比5。许多市售的免疫增强剂 （IRE） 由 2 个组件组成（解决方案 1 和 2，见 材料表）。IRE的专有成分或作用机制尚未披露，但我们先前发现，IRE在固体相绑定检测中减少了抗原和抗体之间的分离常数，表明亲和力的增强至少部分地对IRE6的增强作用负有责任。CDR 与 IRE 的结合产生了一种超高速的西方印迹处理方案，可在不牺牲灵敏度的情况下缩短整个过程时间。

<table>
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Apollo Transparency Film for Laser Printers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any kinds of Laser Printer Transparency Film are fine.</td>
		</tr>
		<tr>
			<td>Azure Imaging System 600</td>
			<td>Azure Biosystems</td>
			<td>Azure 600</td>
			<td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td>
		</tr>
		<tr>
			<td>Can Get Signal Immunoreaction Enhancer Solution</td>
			<td>TOYOBO</td>
			<td>NKB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CARNATION Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from sheep</td>
			<td>GE Healthcare</td>
			<td>NA9310V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from donkey</td>
			<td>GE Healthcare</td>
			<td>NA9340V</td>
			<td></td>
		</tr>
		<tr>
			<td>HYBAID Micro-4</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane, 0.45 &#181;m pore size</td>
			<td>Millipore Sigma</td>
			<td>IPVH304F0</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>KPL LumiGLO Reserve Chemiluminescent Substrate</td>
			<td>seracare</td>
			<td>5430-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#223; actin antibody</td>
			<td>Millipore Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>LICOR</td>
			<td>927-40100</td>
			<td>Blocking Buffer for fluorescent detection</td>
		</tr>
		<tr>
			<td>OXO Salad Spinner</td>
			<td>OXO</td>
			<td>32480V2B</td>
			<td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td>
		</tr>
		<tr>
			<td>Parafilm Sealing Film</td>
			<td>Bemis</td>
			<td>Parafilm M PM996</td>
			<td>semi-transparent flexible film</td>
		</tr>
		<tr>
			<td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Rings to hold 14 ml tube in the center of 50 ml tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in a machine shop</td>
		</tr>
		<tr>
			<td>Rabbit anti-6X His tag antibody</td>
			<td>Abcam</td>
			<td>ab9108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-high-speed western blot using immunoreaction enhancing technology

西方的污点（也称为免疫虫）是生物医学研究的规范方法。它通常用于确定特定蛋白质的相对大小和丰度以及转化后蛋白质的修饰。这种技术具有丰富的历史，由于其简单性，仍然被广泛使用。然而，西方的印迹程序著名的需要几个小时，甚至几天，完成，一个关键的瓶颈是长期的潜伏时间，限制其吞吐量。由于抗体从散装溶液缓慢扩散到膜上的固定抗原，因此需要这些培养步骤：膜附近的抗体浓度远远低于散装浓度。在这里，我们提出了一项创新，通过循环排空和补充抗体溶液 （CDR） 来改善抗原结合，从而显著缩短这些潜伏间隔。我们还利用了增强免疫力的技术来保持检测的灵敏度。CDR方法与商业免疫增强剂相结合，提高了输出信号，大大缩短了抗体潜伏时间。由此产生的超高速西部污点可以在20分钟内完成，而不会失去任何灵敏度。该方法可同时使用化学发光和荧光检测方法应用于西方污点。这种简单的协议使研究人员能够更好地探索许多样本中蛋白质表达的分析。

这个例子说明了CDR方法以及免疫增强技术对西方污点的有效性。静态孵化是在半透明的柔性薄膜上进行的，其中PVDF膜与抗体溶液一起孵育，而CDR孵化则通过含有膜和抗体溶液（电影）的旋转管在混合烤箱中。 图2 显示的抗原和抗体数量分别为西斑化学发光检测所必需的。在静态和 CDR 孵化中，使用 IRE 溶液提高了灵敏度：IRE 将检测 é actin 所需的最低细胞溶解物 （抗原） 数?...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

通过循环排泄和补充 （CDR） 技术与免疫增异剂一起改进抗原抗体结合的动能，开发出一种超高速西方印迹技术。

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

使用场效应生物传感技术探索分子伴侣Hsp90与其客户蛋白激酶Cdc37之间的生物分子相互作用

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

剥离印迹技术：一种冷冻电镜样品制备方法，用于从多层或浓缩生物样品中分离单层

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...