Name: an optimized single-molecule pull-down assay for quantification of protein phosphorylation
Uploaded: 2022-06-06T13:00:00
Description: Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

这项工作得到了美国国立卫生研究院R35GM126934，R01AI153617和R01CA248166的支持。EMB通过阿塞特-伊拉克开发区计划（NIH K12GM088021）和UNM MARC计划（NIH 2T34GM008751-20）提供支持。我们非常感谢使用新墨西哥大学综合癌症中心荧光显微镜共享资源，由NIH P30CA118100支持。我们要感谢安库尔·贾恩博士和哈太基吉普博士，他们最初对SiMPull的开发启发了这项工作。 ES-C现在地址：免疫动力学组，综合癌症免疫学实验室，癌症研究中心，国家癌症研究所，贝塞斯达

1. 盖玻片准备
注意：对于此步骤，需要穿戴个人防护装备（PPE），其中包括双层丁腈手套，安全眼镜或面罩以及实验室外套。
<ol><li>进行食人鱼蚀刻以清除玻璃上的有机碎屑。 注意：食人鱼溶液是一种强氧化剂，与有机材料接触时具有腐蚀性和高度反应性。与有机碎片的反应是放热的，具有潜在的爆炸性。因此，该过程必须在降低窗扇的化学通风橱中进行。...

<ol>
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这里描述的方案经过优化，可以在单一蛋白质水平上定量测量受体磷酸化。对SiMPull方案进行了一些简单但重要的修改，提高了磷酸酪氨酸检测测量的可靠性，包括通过NaBH4 处理减少自发荧光和后固定样品以防止抗体解离。使用绿色通道掩模识别受体位置以计算与抗PY抗体的共定位，还可以通过消除抗体与细胞裂解物的非特异性结合中的潜在伪影来提高测量精度。利用双色成像来检测磷酸?...

膜相关信号传导通过配体诱导的膜受体激活和募集传播信号的下游辅助蛋白的组合进行调整。受体细胞质尾部中关键酪氨酸的磷酸化对于启动信号复合物或信号体1，2的形成至关重要。因此，生物学中的一个重要问题是如何创建和维护磷酸化模式以招募信号伙伴并决定细胞结果。这包括了解受体磷酸化的异质性，无论是在丰度上还是在特定的磷酸酪氨酸模式中，都可以通过决定信号体3，4，5，6，7的组成来提供操纵信号输出的方法。然而，目前询问蛋白质磷酸化的方法存在局限性。蛋白质印迹分析非常适合描述蛋白质磷酸化的趋势，但半定量8，并且不能提供有关系统异质性的信息，因为数千到数百万个受体被平均在一起。虽然蛋白质印迹允许使用磷酸特异性抗体对特定酪氨酸进行探查样品，但它们不能提供有关同一蛋白质内多位磷酸化模式的信息。定量磷酸化蛋白质组学报告了磷酸酪氨酸丰度，但检测多位磷酸化存在局限性，因为感兴趣的残基需要位于酶消化产生的相同肽（通常为7-35个氨基酸）内9，10，11。
为了克服上述限制，单分子下拉（SiMPull）测定已被调整为在单分子水平上量化完整受体的磷酸化状态。SiMPull首先被耆那教等人证明是询问大分子复合物的强大工具。12，13。在SiMPull中，在抗体功能化玻璃盖玻片上对大分子复合物进行免疫沉淀（IP），然后通过单分子显微镜分析蛋白质亚基数和与复合组分12的共IP。Kim等人14的一项修改，称为SiMBlot，是第一个使用SiMPull的变体来分析变性蛋白质的磷酸化。SiMBlot方案依赖于使用NeutrAvidin包被的盖玻片捕获生物素化的细胞表面蛋白，然后用磷酸特异性抗体标记14探测磷酸化。尽管取得了这些进展，但仍需要改进，以使翻译后修饰的定量更加稳健，并适用于更广泛的蛋白质。
本方案描述了一种优化的SiMPull方法，该方法用于量化完整表皮生长因子受体（EGFR）的磷酸化模式，以响应一系列配体条件和致癌突变15。虽然这项工作侧重于EGFR，但这种方法可以应用于任何膜受体和感兴趣的胞质蛋白（POI），其具有高质量的抗体。该协议包括减少样品自发荧光的步骤，需要最小样品体积的样品阵列设计，同时制备多达20个样品，以及优化抗体标记和固定条件。已经开发了用于磷酸化蛋白的单分子检测和定量的数据分析算法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>MTC Bio</td>
			<td>C2000</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl pH 8.0/ 50 mM NaCl</td>
			<td></td>
			<td></td>
			<td>T50 Buffer</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture dish</td>
			<td>CELLTREAT</td>
			<td>229620</td>
			<td>Storage of piranha etched glass/arrays</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td></td>
			<td></td>
			<td>Functionalized Glass storage/ KOH reuse</td>
		</tr>
		<tr>
			<td>50 mM Tris-HCl pH 7.2/150 mM NaCl</td>
			<td></td>
			<td></td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>60 mm Tissue Culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16020</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Acetone (C3H6O)</td>
			<td>Millipore Sigma</td>
			<td>270725</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 NHS Ester</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-20006</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human EGFR (External Domain) &#8211; Biotin</td>
			<td>Leinco Technologies, Inc</td>
			<td>E101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p-Tyr Antibody (PY99) Alexa Fluor 647</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7020 AF647</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath-sonicator</td>
			<td>Branson</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Gold Biotechnology</td>
			<td>A-420-1</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Buchner funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Millipore Sigma</td>
			<td>C4901</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Bioworld</td>
			<td>30900017-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Filtering Flask</td>
			<td>Fisher Scientific</td>
			<td>S15464</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>WHEATON</td>
			<td>900470</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24 x 60 #1.5</td>
			<td>Electron Microscopy Sciences</td>
			<td>63793</td>
			<td></td>
		</tr>
		<tr>
			<td>DipImage</td>
			<td></td>
			<td></td>
			<td>https://diplib.org/</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Caisson Labs</td>
			<td>DML19-500</td>
			<td></td>
		</tr>
		<tr>
			<td>emCCD camera</td>
			<td>Andor iXon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Optima</td>
			<td>Bio-Techne</td>
			<td>S12450H</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>Fusion 360 software</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin G418 Disulfate</td>
			<td>Caisson Labs</td>
			<td>G030-5GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid (CH3COOH)</td>
			<td>JT Baker</td>
			<td>JTB-9526-01</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Glass serological pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Stir Rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-(+)-Glucose)</td>
			<td>Millipore Sigma</td>
			<td>D9434</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Halt Phosphotase and Protease Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>78446</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (HCl)</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (3%)</td>
			<td></td>
			<td>HX0645</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (H2O2) (30%)</td>
			<td>EMD Millipore</td>
			<td>HX0635-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (NP-40)</td>
			<td>Sigma Aldrich</td>
			<td>I8896</td>
			<td>Lysis Buffer Component</td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol 518F immersion oil</td>
			<td>Zeiss</td>
			<td>444960-0000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>in-house vacuum line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnessium Chloride Hexahydrate (MgCl2-6H2O)</td>
			<td>MPBio</td>
			<td>2191421</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td>Curve Fitting Toolbox, Parallel Computing Toolbox, and Statistics and Machine Learning toolbox</td>
		</tr>
		<tr>
			<td>Methanol (CH3OH)</td>
			<td>IBIS Scientific</td>
			<td>MX0486-1</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix-n-Stain CF Dye Antibody Labeling Kits</td>
			<td>Biotium</td>
			<td>92245</td>
			<td>Suggested conjugation kit</td>
		</tr>
		<tr>
			<td>mPEG</td>
			<td>Laysan Bio</td>
			<td>mPEG-succinimidyl valerate, MW 5,000</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(2-aminoethyl)-3-aminopropyltrimethoxysilane</td>
			<td>UCT United Chemical</td>
			<td>A0700</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Nanogrid</td>
			<td>Miraloma Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeutrAvidin Biotin Binding Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen (compressed gas)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NVIDIA GPU with CUDA</td>
			<td></td>
			<td></td>
			<td>Look for compatibility at https://www.mathworks.com/help/parallel-computing/gpu-support-by-release.html</td>
		</tr>
		<tr>
			<td>Olympus iX71 Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M Sealing Film</td>
			<td>The Lab Depot</td>
			<td>HS234526C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>Caisson Labs</td>
			<td>PBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-200 Analog Hot Plate</td>
			<td>Corning</td>
			<td>6795-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb</td>
			<td>Cell Signaling Technology</td>
			<td>2236BF</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Millipore Sigma</td>
			<td>529552</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Millipore Sigma</td>
			<td>1050330500</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Premium PLA Filament, 1.75 mm diameter</td>
			<td>Raise 3D</td>
			<td>PMS:2035U/RAL:3028</td>
			<td>Printing temperature range: 205-235 &#176;C</td>
		</tr>
		<tr>
			<td>Pro2 3D printer</td>
			<td>Raise 3D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 1 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 100 mL storage bottles</td>
			<td>Corning</td>
			<td>1395-100</td>
			<td>CH3OH/C3H6O reuse</td>
		</tr>
		<tr>
			<td>Pyrex 250 mL beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 4 L beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-view Image Splitter</td>
			<td>Photometrics</td>
			<td>Model QV2</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>RevCount Cell Counters, EVE Cell Counting Slides</td>
			<td>VWR</td>
			<td>10027-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Semrock emission filters: blue (445/45 nm), green (525/45 nm), red (600/37 nm), far-red (685/40 nm)</td>
			<td>Semrock</td>
			<td>LF405/488/561/635-4X4M-B-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>smite single molecule analysis package</td>
			<td></td>
			<td></td>
			<td>https://github.com/LidkeLab/smite.git</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sodium Borohydride (NaBH4)</td>
			<td>Millipore Sigma</td>
			<td>452874</td>
			<td>Tyrode&#39;s Buffer Component</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Millipore Sigma</td>
			<td>S9625</td>
			<td>Activate by successive heat and pH cycling</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>VWR</td>
			<td>BDH3247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Millipore Sigma</td>
			<td>S6508</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (H2SO4)</td>
			<td>Millipore Sigma</td>
			<td>258105</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7279</td>
			<td>multi-fluorescent beads</td>
		</tr>
		<tr>
			<td>Tris (Trizma) base</td>
			<td>Millipore Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300120</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized single-molecule pull-down assay for quantification of protein phosphorylation

磷酸化是一种必要的翻译后修饰，可调节蛋白质功能并指导细胞信号传导结果。目前测量蛋白质磷酸化的方法不能保持单个蛋白质磷酸化的异质性。单分子下拉（SiMPull）测定是通过在玻璃盖玻片 上免疫沉淀 蛋白质然后进行单分子成像来研究大分子复合物的组成。目前的技术是对SiMPull的改编，可在单分子水平上对全长膜受体的磷酸化状态进行强有力的定量。以这种方式对数千个个体受体进行成像可以量化蛋白质磷酸化模式。本实验方案详细介绍了优化的SiMPull程序，从样品制备到成像。玻璃制备和抗体固定方案的优化进一步提高了数据质量。目前的方案为单分子数据分析提供了代码，该数据分析计算样品中磷酸化的受体的比例。虽然这项工作的重点是表皮生长因子受体（EGFR）的磷酸化，但该方案可以推广到其他膜受体和胞质信号分子。

图 1A显示了一幅描绘SiMPull过程的卡通。使用新链阿维丁作为生物素化抗EGFR抗体的锚点，从总蛋白裂解物中捕获EGFR-GFP，盖玻片被功能化。洗去未结合的蛋白质后，磷酸化的受体用抗磷酸酪氨酸（anti-PY）抗体15标记。 图1B 显示了疏水阵列的图像，其中可以制备多个样品并在同一盖玻片上成像。该样品架的一个优点是需要~10 μL的最小样?...

Watch this Scientific Journal Video about An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation at JoVE.com

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

本方案描述了样品制备和数据分析，以使用改进的单分子下拉（SiMPull）测定来量化蛋白质磷酸化。

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

该SiMPull方案通过改善抗体标记和固定条件，减少绿色通道中发现的自发荧光并提供强大的单分子分析算法，实现蛋白质磷酸化的定量。SiMPull的主要优点是它允许我们询问单个完整蛋白质的磷酸化状态。通过这种方法，我们可以获得通过传统技术无法获得的新信息，例如蛋白质印迹和质谱分析。我们有一个研究团队来帮助演示该协议。首先，朱利安·罗霍将展示如何皮兰哈蚀刻盖玻片。然后，雷切尔·格拉坦将展示盖板玻璃的涂层步骤。伊丽莎白·贝利将展示如何绘制阵列和图像样本。首先，食人鱼蚀刻盖子滑，每五分钟轻轻搅拌一次反应烧杯，持续30分钟。通过逐渐添加弱碱来中和食人鱼溶液。用玻璃棒将蚀刻的盖子滑入Buchner漏斗中，然后在双蒸馏水中冲洗五分钟。然后将盖玻片放入玻璃Coplin罐中，并用甲醇覆盖。用密封膜密封盖子，并浸泡超声处理10分钟。超声处理后，将甲醇倒入玻璃储存瓶中。用丙酮重复此步骤。用双蒸馏水在科普林罐中冲洗盖板三次。从盖玻片中沥干水，并通过在本生燃烧器的火焰中挥手将其干燥。将盖玻片放入干燥的科普林罐中。然后将氨基硅烷溶液加入Coplin罐中，进行覆盖滑移氨基硅烷化。盖上并涂上密封膜以保护避光。用甲醇冲洗盖玻片，并丢弃用过的溶液。再次，用双蒸馏水冲洗盖板滑三次，每次两分钟。轻拍掉多余的水分，完全风干 10 分钟。接下来，使用疏水性屏障笔绘制网格阵列。在封面滑轨上标记标识符以标识正确的方向。墨水干燥后，将盖玻片放入加湿室中。将153毫克mPEG和3.9毫克生物素-PEG重悬于609微升10毫摩尔碳酸氢钠和涡旋中。在室温下以10， 000 x g 离心一分钟以除去气泡。每平方应用10至15微升该溶液，以完全覆盖阵列而不会溢出。将盖子滑入潮湿室，在室温下在黑暗中储存三到四个小时。然后将盖玻片依次浸入三个装满双蒸馏水的烧杯中10秒钟，将其清洗。使用氮气清除盖玻片上的所有水分。将盖玻片背靠背存放在装有氮气的50毫升锥形管中，并用密封膜密封。用密封膜包裹管子，并将其置于零下20摄氏度。从冰箱中取出生物素-PEG功能化阵列，并将其平衡至室温。将盖玻片放在阵列朝上，放在衬有密封膜的100毫米组织培养皿上。在室温下，将阵列的每个正方形与每毫升10毫克的硼氢化钠在PBS中孵育四分钟。用 PBS 洗涤三次。接下来在T50中孵育每毫升0.2毫克的纽曲阿维丁五分钟，然后用T50 BSA洗涤三次。在T50 BSA中重复每毫升两微克的生物素化POI特异性抗体孵育10分钟，然后洗涤。首先，通过移液解冻混合裂解物。将一微升裂解物稀释成100微升冰冷的T50 BSA PPI。将裂解物在阵列上孵育10分钟，然后洗涤。在冰冷的T50 BSA PPI中制备AF647偶联抗磷酸酪氨酸抗体，并在阵列上孵育一小时。在物镜上滴一滴油。将纳米网格放在舞台上进行成像。使用透射的白光，聚焦在网格图案上。获取网格的一系列20张图像，确保像素不饱和，并将图像系列保存为基准。散焦纳米网格以创建通风图案，获取一系列20张图像以进行增益校准，并将图像保存为增益。然后获取一系列20张图像，通过阻挡所有光线到相机进行偏移，并保存图像背景。要获得简单的图像，首先，清洁油物镜并在物镜上沉积额外的油。将盖玻片阵列固定在显微镜载物台上。采集每个样品的图像，首先在远红色通道中，然后是较低波长的荧光团。每30至45分钟检查一次缓冲液水平，并根据需要补充。使用这种技术，从总蛋白裂解物中捕获EGFR-GFP。疏水性油墨的自发荧光被用作寻找样品焦平面的指南。原始数据图像是在相机芯片上分离的光谱通道采集的。进一步检查了绿色和远红色通道的叠加层以进行数据采集。对从纳米网格获取的图像进行通道配准。基准图像的叠加显示配准不完整。对齐是通过在远红色和绿色通道坐标上应用局部加权均值变换来完成的，该变换用于注册随后的SiMPull数据。来自GFP和AF647通道的背景光子计数以上的单个发射器被框住，并执行高斯定位以识别位置。EGFR-GFP在F647中的共定位是为了鉴定磷酸化受体。共定位的百分比用于确定磷酸化受体的比例。当食人鱼蚀刻玻璃用硼氢化钠处理时，背景检测减少。在裂解物中观察到EGFR-GFP的强健结合，具有最小的非特异性PY99-AF647结合，表明使用该技术保留了表面官能化。SiMPull提供有关蛋白质磷酸化的信息，可以解决广泛的自信号问题。这可以增强我们对正常和疾病状态下信号传导的理解。

Research

JoVE Journal

Biochemistry

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

Oligopeptide Competition Assay for Phosphorylation Site Determination

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

大肠癌细胞中磷酸化和微管结合与 Tau 蛋白定位的测定

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...