Name: a semi-quantitative drug affinity responsive target stability (darts) assay for studying rapamycin/mtor interaction
Uploaded: 2019-08-27T14:00:00
Description: Watch this Scientific Journal Video about A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction at JoVE.com

这项工作部分得到了NIH研究资助R01NS103931、R01AR062207、R01AR061484和国防部研究补助金W81XWH-16-1-0482的支持。

1. 收集和酶细胞
<ol><li>使用Dulbeco的改性Eagle培养基（DMEM）与10%胎儿牛血清、2 mM谷氨酰胺和1%抗生素一起培育293T细胞。在 37°C 下孵育培养物，在 5% CO2下孵育。 注：细胞的生长状态可能会影响后续实验的稳定性。</li>
	<li>扩大培养细胞，直到达到80\u201290%汇合。</li>
	<li>将345 μL的细胞溶化试剂（见材料表）与25μL的20x蛋白酶抑制剂鸡尾酒、25μL的1M氟化钠、50μL的100mβ-糖酸磷?...

<ol>
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DARTS 允许利用蛋白质结合对降解的保护作用来识别小分子靶点。DARTS不需要任何化学修饰或小分子的固定26。这允许使用小分子来确定其直接结合蛋白靶点。经典DARTS方法的标准评估标准包括凝胶染色、质谱和西印12、13。经典方法还提到这些数据可以定量分析，但没有提供这样的示例。在这里，我们使用蜂窝热移变测定（CETSA）的原理?...

识别小分子靶蛋白对于机械性理解和开发潜在的治疗药物1，2，3至关重要。亲和色谱作为识别小分子靶蛋白的经典方法，取得了良好的效果4，5。然而，这种方法有局限性，因为小分子的化学修饰往往导致结合特异性或亲和力的减少或改变。为了克服这些限制，最近已经开发并应用了几种新的策略来识别小分子目标，而不需要对小分子进行化学修饰。这些用于无标签小分子目标识别的直接方法包括药物亲和力响应靶稳定（DARTS）6、蛋白质从氧化率（SPROX）7的稳定性、细胞热移位测定（CETSA）8 ，9和热蛋白酶分析 （TPP）10。这些方法是非常有利的，因为它们使用天然的，未经修饰的小分子，并仅依靠直接结合相互作用，以找到目标蛋白质11。
在这些新方法中，DARTS是一种相对简单的方法，大多数实验室都可以轻松采用。DARTS 取决于配体结合蛋白相对于未结合的蛋白质表现出对酶降解的经修饰的易感性的概念。通过液相色谱-质谱法（LC-MS/MS）检查SDS-PAGE凝胶中改变的带状，可以检测出新的靶蛋白。该方法已成功实施，用于识别以前未知的天然产品和药物目标 14、15、16、17、18、19.它作为筛选或验证化合物与特定蛋白质20、21结合的手段也非常强大。在这项研究中，我们通过监测小分子的蛋白质稳定性变化和识别蛋白质配体结合亲和力，对实验进行了改进。我们以mTOR-雷帕霉素相互作用为例来演示我们的方法。

<table>
	<tbody>
		<tr>
			<td>100X Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>Dilute to 20X with ultrapure water</td>
		</tr>
		<tr>
			<td>293T cell line</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>DMEM medium with 10% FBS</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Thermo Fisher</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 Staining Solution</td>
			<td>Bio-Rad</td>
			<td>1610436</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Version 6.0</td>
			<td>statistical analysis and drawing software</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>Version 1.52</td>
			<td>image processing and analysis software</td>
		</tr>
		<tr>
			<td>M-PER Cell Lysis Reagent</td>
			<td>Thermo Fisher</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>R21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche</td>
			<td>PRON-RO</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>S7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>221368</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>adjust to pH 8.0</td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

a semi-quantitative drug affinity responsive target stability (darts) assay for studying rapamycin/mtor interaction

药物亲和力响应目标稳定性 （DARTS） 是检测新型小分子蛋白靶点的有力方法。它可用于验证已知的小分子-蛋白质相互作用，并找到天然产物的潜在蛋白质靶点。与其他方法相比，DARTS 使用原生、未经修饰的小分子，操作简单易行。在这项研究中，我们通过监测蛋白质稳定性的变化和估计蛋白质-配体相互作用的亲和力，进一步提高了DARTS实验的数据分析能力。蛋白质-配体相互作用可以绘制成两条曲线：蛋白质解解曲线和剂量依赖曲线。我们使用 mTOR-拉帕霉素相互作用作为建立我们协议的典范案例。从蛋白溶酶曲线中，我们看到，通过蛋白酶抑制mTOR的蛋白解酶被雷帕霉素的存在所抑制。剂量依赖曲线使我们能够估计雷帕霉素和 mTOR 的结合亲和力。该方法可能是准确识别新型靶蛋白和优化药物靶向的一种强大而简单的方法。

实验的流程图如图1所示。库马西蓝色染色的结果如图2所示。用小分子孵育可以保护蛋白解。发现三个带，似乎通过孵化与雷帕霉素在车辆控制下孵化。蛋白解曲线实验的预期结果如图3所示。作为原理证明，我们检查了经过充分研究的蛋白质mTOR，这是药物雷帕霉素25的目标。西方印迹说明了mTOR蛋白在低蛋白酶中...

Watch this Scientific Journal Video about A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction at JoVE.com

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

在这项研究中，我们通过监测蛋白质稳定性的变化和估计蛋白质-配体相互作用的亲和力，增强了DARTS实验的数据分析能力。相互作用可以绘制成两条曲线：蛋白质解解曲线和剂量依赖曲线。我们使用 mTOR-拉帕霉素相互作用作为示例案例。

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性 （DARTS） 测定

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

药物亲和力反应目标稳定性，DARTS，是检测新型小分子蛋白靶点的一种稳健方法。它可用于验证已知的小分子蛋白相互作用，并找到天然产品的潜在蛋白质靶点。在这项研究中，我们通过监测蛋白质稳定性的变化和估计蛋白质配体相互作用的亲和力，进一步增强了DARTS实验的数据分析能力。蛋白质配体相互作用可以根据两条曲线绘制，一条蛋白解曲线和一条剂量依赖曲线。我们使用 mTOR-rapamycin 交互作为建立协议的典范案例。使用DMEM生长293T细胞，使用10%的胎儿牛血清、两毫升谷氨酰胺和1%抗生素。在37摄氏度的二氧化碳下孵育。当培养达到80-90%汇合时，用冷PBS清洗细胞两次。使用细胞刮刀将细胞收集到适当数量的冷细胞解解缓冲液中，将解菌细胞转移到1.5毫米管中。倒置混合解液缓冲液和解菌细胞，并在冰上孵化管子10分钟。在4摄氏度下，将管子以18，000倍g离心10分钟。将上一液转移到新的1.5毫升管中，并在冰上冷藏。执行 BCA 测定以近似于 lysate 的蛋白质浓度，并基于蛋白质浓度计算蛋白酶的稀释。首先，将酸盐转移到两个1.5毫升管中，每个管99微升。在每个糖酸盐的一个分体中加入一微升的小分子库存溶液，并在室温下通过摇动孵育两个管30至60分钟。在冰上，在1x TNC中建立新鲜解冻的蛋白酶溶液的连续稀释。与小分子孵育后，将每个等分分成5个管中的20微升样品。为了确保相同的消化时间，每30秒的特定间隔，在样品中加入两个微升的已准备前酶溶液。对于一个控制组，添加两个微升的 TNC 缓冲区。5至20分钟后，通过每隔30秒加入两微升冷20x蛋白酶抑制剂鸡尾酒，停止6管的消化。混合好，在冰上孵育10分钟。用 6 微升 5x SDS-PAGE 加载缓冲液稀释每个样品，并在 70 摄氏度的水浴中将样品煮沸 10 分钟。现在，要执行西式印迹，将等量的蛋白质加载到8%SDS-PAGE凝胶的井中，以及适当的分子量标记物中。在 80 伏电压下运行凝胶 30 分钟，然后将电压调整为 120 伏，然后继续运行 1 到 2 小时。验证小分子能直接与潜在的靶蛋白结合。在此协议中，与小分子的孵育证实了对蛋白细胞分解的保护。发现三个带子在车辆控制上通过使用拉帕霉素进行孵育而受到保护。西方印迹表明mTOR蛋白在低前酶与蛋白质比率中的存在，以及其减少和损失与增加的比例。由蛋白酶对mTOR的蛋白酶的蛋白酶明显抑制，拉帕霉素的存在和拉帕霉素的加入在蛋白酶曲线上产生了明显的变化。拉帕霉素剂量依赖性地提高了mTOR的水平，表明mTOR与拉帕霉素治疗的稳定性上升。目标蛋白带强度的定量表明mTOR是拉帕霉素的目标蛋白。为了获得最佳的步进效果，本实验可能需要重复多次，以获得适当的蛋白比前鼻作用。

Research

JoVE Journal

Biochemistry

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential Scanning Calorimetry ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性 （DARTS） 测定

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性（DARTS）测定