Name: calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software
Uploaded: 2018-07-17T15:30:00.000+00:00
Duration: 8 min 22 s
Description: Watch this Scientific Journal Video about Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software a

这项工作得到了威康信托基金 105278/Z/14/2 对 R.N. 的支持。威康人类遗传学信托中心由威康信托核心奖 203852/Z/16/2 资助。校准小组的工作由英国癌症研究 (C20724/A14414) 和欧洲研究理事会根据欧洲联盟2020研究和创新方案赠款647278提供支持。

1. FKBP12 F36V-mVenus 纯化
<ol><li>转换 (DE3) pLysS 细胞与 pET22b 向量包含 monomerized 人类 FKBP12F36V12和 N 终端 His6 和 mVenus 标签 (向量可根据要求提供)。板细胞到 LB 琼脂补充50µg/毫升氨苄西林和34µg/毫升氯霉素。</li>
	<li>转移转化成的菌落为100毫升磅起始培养和增长 16-20 小时在37°c 与震动。</li>
	<li>稀密启动培养 (OD600 > 1) 1:100 在 LB 介质 (2 x 500 毫升批次) 和成长为 2-3 小时到 OD600 = 0.6-0.8 (37 °c, 200 rpm)。</li>
	<li>冰上的清凉文化。诱导与250µM IPTG 和增长 16-20 小时在21°c, 200 rpm。</li>
	<li>通过离心 2000 x g 20 分钟来收获细胞。</li>
	<li>并用重悬颗粒在40毫升的 IMAC 缓冲器 A (20 毫米磷酸钠 7.5, 500 毫米氯化钠, 3 毫米咪唑, 1 毫米β-巯基乙醇) 补充无 EDTA 蛋白酶抑制剂 (1 片剂每细胞颗粒)。</li>
	<li>油脂实验细胞 (500 瓦特, 20 赫, 40% 振幅, 9 s 在, 十一年代关闭为15分钟) 在冰和收获可溶性材料通过离心在 2万 x g。</li>
	<li>将可溶性裂解物转移到锥形瓶中, 加入2毫升树脂 (见材料表)。孵育1小时, 105 rpm 旋转 注: 镍琼脂糖也可用于此 IMAC 步骤。</li>
	<li>收获树脂和洗涤与250毫升 imac 缓冲器 A 跟随500毫升 imac 缓冲 B (20 毫米磷酸钠 pH 值 7.5, 500 毫米氯化钠, 7 毫米咪唑, 1 毫米β巯基乙醇)。 注: 如果使用镍琼脂糖树脂, 增加到50毫米咪唑。</li>
	<li>洗脱 His6-tagged 蛋白使用 IMAC 缓冲 C (20 毫米磷酸钠 pH 值 7.5, 500 毫米氯化钠, 300 毫米咪唑, 1 毫米β-巯基乙醇)。</li>
	<li>注入一个尺寸排除柱 (见材料表) 平衡在10毫米 HEPES pH 值 7.5, 150 毫米氯化钠, 1 毫米的德勤。FKBP12F36V 在我们使用的柱子上有87.71 毫升的峰值洗脱。</li>
	<li>通过 SDS 页和水池评估纯度, 并根据需要进行浓缩。</li>
</ol>2. 多井板阵列的制备
<ol><li>从-80 摄氏度解冻纯化的 FKBP12F36V (或标记的感兴趣的蛋白质)。</li>
	<li>制备100纳米纯化 FKBP12F36V (中、10毫米 HEPES pH 7.5、150毫米氯化钠、1毫米) 的溶液。油脂实验和离心机 (快速旋转 1.3万 rpm), 以防止骨料的形成。</li>
	<li>吸管 100-200 µL 进入一个8井观察室与玻璃底部。</li>
	<li>将 BB dimerizer 添加到最终浓度为10、20、40、80、100、150、300和 500 nM12、13。</li>
	<li>作为参考, 单独准备100纳米 mVenus 的溶液来评估潜在的聚集和沉淀效应, 并恢复具有相同采集设置的单体的亮度值。</li>
</ol>3. 无标定共焦采集
<ol><li>启动共焦系统 (图 1)。任何光扫描显微镜共聚焦系统配备数字探测器或具有良好特征的模拟探测器8, 并能够保持恒定的停留时间为每一个像素获得将工作。</li>
	<li>设置励磁光束路径:<ol>
<li>打开 514 nm 激光, 并设置它在 20-100 nW 功率在目标的出口 (为 FKBP12F36V-mVenus)。</li>
		<li>选择63X1.4NA 目标或为 FCS 设计的衣领校正水浸泡目标。</li>
		<li>打开一个液压、APD 或校准的 PMT 探测器。能够进行光子计数的探测器是可取的, 就像在这种情况下, 计算S、偏移和σ02是不必要的。</li>
		<li>从 520-560 nm 中选择发射窗口</li>
		<li>设置针孔在1通风单位为相应的发射 ~ 545 nm。</li>
		<li>将捕获模式设置为 16 x 16 像素</li>
		<li>设置像素停留时间t居住, 以满足t框> > tD > > t驻留, 其中D是扩散蛋白的停留时间和t帧是帧速率。这对应于设置停留时间到 ~ 13 µs。 注意: 一些商业制造商的扫描仪没有保持每个像素常数的驻留时间。这种恒定是方法工作的关键。</li>
		<li>将像素大小设置为 ~ 120 nm。</li>
		<li>选择xyt捕获模式, 并选择每个获取和良好 (例如 5000) 获得的帧数。</li>
		<li>如果系统配备了高通量模式, 请介绍每个井的坐标和每个井的购置数量, 以实现自动化过程。 注意: 如果使用浸入式目标, 请小心确保水分配器的存在。</li>
		<li>如果系统配备了灌注系统, 加载 BB 溶液和程序的灌注开始右-5000 帧后, 以评估二聚化的动力学, 而获得例如, 1万图像。</li>
	</ol>
</li>
	<li>如果使用为 FCS 设计的衣领校正水浸泡目标, 请在油浸泡目标/水中加入一滴油。</li>
	<li>登上8井观察室进入阶段。</li>
	<li>选择正确的井, 重点放在解决方案上。 注意: 重要: 避免靠近底部玻璃, 避免反射和散射。在深入解决方案时, 请断开自动焦点选项。</li>
	<li>开始获取并以 TIFF 格式保存生成的图像堆栈。</li>
</ol>4. 使用 R 封装的 Detrend 和亮度分析 nandb
<ol><li>作为预处理质量检查, 请使用 ImageJ14查看图像并恢复强度配置文件, 如图 2a所示。这是很有用的, 以确定是否有太多的漂白已经发生。如果漂白太多, 数据不适合进一步分析。 注意: ImageJ 也可以用来将图像从商业格式转换为 TIFF。下面描述的 nandb 软件只能用于 TIFF 文件。</li>
	<li>下载并安装 R 和 RStudio15,16。最好先下载并安装 R, 然后再 RStudio。 注意: 下面是如何使用 nandb R 包的说明。对于 R 语言的知识不需要使用 nandb, 但是它会使生活更轻松。</li>
	<li>安装 nandb 包。<ol>
<li>打开 RStudio 并在控制台中, 键入安装. 软件包 ("nandb") 并等待安装。</li>
	</ol>
</li>
	<li>了解 nandb<ol>
<li>查看手册17。</li>
		<li>查看各种功能的内置 RStudio 帮助。最可能使用的函数将是亮度 ()。通过键入来查看此函数的帮助文件吗？亮度 () 在控制台上。</li>
	</ol>
</li>
	<li>计算亮度<ol>
<li>假设在桌面上有一个名为 img001 的图像文件 (请注意, "nandb" 只适用于 TIFF 文件)。你可以计算该图像的亮度: b <-亮度 ("〜/桌面/img001", 头 = "自动")<ol>
<li>这将图像的亮度分配给 R 中的变量 b。头 = "自动" 确保图像在亮度计算之前正确去趋势。从这里最常见的事情是计算图像的平均亮度或中值。你可以通过键入平均值 (b) 或中位 (b) 来做到这一点。你也可以写亮度图像到桌面使用 ijtiff:: write_tif (b, "〜/台式机/whatever_img_name")</li>
		</ol>
</li>
		<li>比方说, 一个文件夹中的图像 images_folder 在桌面上, 一个需要计算这些图像的亮度, 并将亮度图像作为 TIFF 文件写入。然后看 brightness_folder ()。此函数同时处理整个文件夹: brightness_folder ("〜/桌面/images_folder", 头 = "自动") 这对于那些有他们喜欢的软件的人来说特别好, 因为所有的文件都是在一个命令中处理的, 然后你可以在他们选择的软件中继续处理输出亮度 TIFF 图像, ImageJ14, Python 或其他东西。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

N & B 是一种检测 multimerization 的技术, 使用商用光扫描共聚焦显微镜配备数字探测器。与单点 FCS、FCCS 和 smFRET 相比, 该方法具有很好的吸引力, 因为它是无标定的, 亮度计算简单, 浓度独立6。然而, 在执行亮度计算9之前, 纠正漂白和长期强度波动是很重要的;如果 detrending 被忽略或不正确地执行, 由于漂白和其他工件的亮度轻微增加可能被误解为寡聚状态的变化 (如...

蛋白质-蛋白质相互作用I n-体外
传统上, 晶体学和核磁共振实验结合低温电子显微镜 (cryoEM) 是选择准确描述蛋白质三维结构的技术, 并推断其功能审议他们的高分辨率结构细节。然而, 蛋白质不是静止的结构, 可以经受各种构象变化和时间和空间的振动。这就是为什么来自晶体或 CryoEM 数据的结构信息需要辅以其他技术 (例如,分子动力学模拟和单分子技术): 蛋白质的功能与其构象有关。更改和交互, 并且此信息不存在于静态结构中。为了探索分子内动力学, 基于单分子福斯特共振能量转移 (smFRET) 的技术是非常有效的1。这些方法能够评估复杂介质中分子的不同亚群。这是非常重要的, 因为这些变化是迅速的, 并发生在获取数据 (即纳秒到第二个范围)。
通常使用两种主要方法来检测和量化这些变化: 溶液中的蛋白质和表面固定化。对于分子间相互作用的检测, 特别是配体诱导的二聚化过程, smFRET 并不总是最佳的工具。的确, 烦恼不仅依赖于距离 (≈10 nm), 而且还取决于两个偶极子 (施主和受体, χ2) 的方向和供体发射与受体的吸收光谱2的重叠, 但也许这最后的条件是少重要的是, 如果实验家可以选择正确的焦虑夫妇。smFRET 探测二聚化的一个特殊缺点来自于对感兴趣的蛋白质的标记: 对于异性 smFRET, 二聚化只能检测到 50% (即,异种焦虑只能检测到捐赠者受体和受体-捐赠者的脂肪酸, 但不是捐助者或受体-受体, 这是其他50% 的脂肪酸)。利用荧光相关光谱学 (FCS) 和衍生物 (FCCS等3) 来确定蛋白质的扩散常数和体外结合常数是另一种选择。这些方法无法完全量化二聚化, 就像在 FCS 一测量浓度和扩散, 扩散粒子的半径和扩散系数非常依赖于分子量;例如, 分子量的10倍增加只意味着扩散系数4的2.15 倍变化。在双色 FCS 或 FCCS 的情况下, 只有50% 的脂肪酸将被视为与上述相同的原因。在体外和体内检测二聚化的最实用和定量的方法是5 、数字和亮度 (N & B)6。鉴于人类的焦虑需要特定的仪器恢复的各向异性值 (即光学元件/分析仪, 以恢复平行和垂直极化), N & B 在这里提出作为一个有利的技术, 以检测蛋白质二聚化和聚合。它可以在体外和体内使用商业设置。
数字和亮度
N & B 最近被审查了7。该评价的重点是技术在活细胞中的应用。这是值得在这里重现的数学形式主义, 因为这些等式将应用于收集的数据体外。首先, 有必要定义一些术语和数学量:
<ul><li>实体是一组被绑定在一起的分子。</li>
	<li>一个实体的亮度ε是每单位时间 (每帧) 发出的光子数。</li>
	<li>
n是存在的实体数。</li>
	<li>对于图像序列中的给定像素, 是其平均强度, σ2是其强度的方差。</li>
</ul>然后, 用光子计数探测器和假设移动实体, 没有背景,
<img alt="Equation 1" src="/files/ftp_upload/58157/58157eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/58157/58157eq2.jpg">
其中N是明显的数字, B是明显的亮度。这会导致
<img alt="Equation 3" src="/files/ftp_upload/58157/58157eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/58157/58157eq4.jpg">
Dalal等。8表明, 与模拟设备, 你需要三更正术语: S 因素, 背景偏移, 和读数噪声σ02。然后, 再次假设移动实体,
<img alt="Equation 5" src="/files/ftp_upload/58157/58157eq5.jpg"> <img alt="Equation 6" src="/files/ftp_upload/58157/58157eq6.jpg">
给
<img alt="Equation 7" src="/files/ftp_upload/58157/58157eq7.jpg"> <img alt="Equation 8" src="/files/ftp_upload/58157/58157eq8.jpg">
请注意, 上述等式是不同的, 给在 Dalal等。8和随后的审查。7在 Dalal等, 分母中的S被省略了由于一个错字和这个错误被复制在审查。上面的等式是正确的。测量S, 偏移和σ02的指示-连同他们的意思解释-是由 Dalal等人给出的。8
亮度ε是成正比的寡聚状态的扩散实体: ε将是两倍大的脂肪酸, 因为它是对单体, 三倍大的三聚体, 因为它是为单体, 两倍大的 hexamers, 因为它是三聚体等。这样, 测量亮度ε, 你可以量化任何类型的 multimerization。
如果存在寡聚状态的混合物, 则数字和亮度无法恢复所呈现的单个寡聚状态。这是技术的局限。
Detrend 算法与 nandb 软件
对漂白的纠正的重要性以前曾被强调过9。漂白在光镜实验中不可避免地出现时间失效模式;在活细胞和体外。在文献中已经描述了许多方法, 以纠正漂白7。detrending 参数T的自动选择指数滤波技术是目前最好的方法。它集成到免费的开源软件 nandb9中。实际上, 需要用户手动选择其 detrending 参数的软件可能会导致错误的结果, 因为此参数选择可能是任意的和不正确的。自动算法检查数据并确定相应的参数, 不需要用户干预9。即使使用平滑参数的最佳选择, detrending 也有其局限性, 并且仅在小于25% 的漂白百分比下工作, 如模拟9所示。有趣的是, 当使用自动 detrending 例程时, 它的精确度是这样的, 你可以使用低亮度值 (即使是B < 1.01), 因此低强度, 仍然是精确到量化的二聚化。
漂白还导致另一个问题: photobleached 显影存在于 multimer 复杂中。这使例如,当三聚体中的三个单位中的一个是非荧光的时候, 三聚体就像一个二聚体。宾虚和米勒10显示了如何纠正这一点, 这一修正也强调在随后的审查7。nandb 软件包括此更正9。
FKBP12F36V系统
FKBP12F36V 是一种不自然 oligomerize 的蛋白质, 但 dimerize 在 AP20187 药物 (俗称 BB dimerizing 配体)11、12的加入后才知道。这使得它成为一个理想的数字和亮度测试用例: 用标记的 FKBP12F36V, 寡聚状态的加倍应该观察在加法 BB。

<table><tbody><tr><td>RosettaTM (DE3) pLysS cells</td><td>Novagen</td><td>70956-3</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>PubChem Substance ID 329824407</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma Aldrich</td><td>PubChem Substance ID: 24892250</td><td></td></tr><tr><td>LB starter culture</td><td>QIAGEN</td><td></td><td></td></tr><tr><td>LB medium</td><td>QIAGEN</td><td>https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif</td><td></td></tr><tr><td>IPTG</td><td>Sigma Aldrich</td><td>PubChem Substance ID 329815691</td><td></td></tr><tr><td>IMAC buffer</td><td>Medicago</td><td>09-1010-10</td><td></td></tr><tr><td>EDTA-free protease inhibitors&amp;nbsp;</td><td>Sigma Aldrich</td><td>11873580001</td><td></td></tr><tr><td>TALON resin</td><td>Clonetech</td><td></td><td></td></tr><tr><td>Nickel sepharose</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>S200 16/60 column</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Glass bottom 8 well observation dish</td><td>Ibidi</td><td>80827</td><td></td></tr></tbody></table>

calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software

数字和亮度是一种无标定荧光涨落光谱 (FFS) 技术, 用于检测蛋白质的分子齐聚。它可以使用传统的共焦显微镜配备数字探测器。在体外使用该技术的一项协议通过一个使用案例显示, 在该用例中, 数字和亮度可以准确量化 mVenus 标记的 FKBP12F36V 在 dimerizing 药物 AP20187 添加之前和之后的寡聚状态。讨论了使用正确的显微采集参数和正确的数据预处理和分析方法的重要性。尤其强调了选择漂白校正的重要性。这种廉价的方法可以用来研究蛋白质与蛋白质之间的相互作用, 在许多生物环境。

Detrending 和单体亮度
获取数据后, 可以开始亮度计算, 以确定解决方案中感兴趣的蛋白质的寡聚状态。即使漂白在溶液中的作用可能不会像体内那样剧烈, 但强度痕迹仍可能没有固定的平均值, 可能是由于与荧光有关的物理效应,18但背后的原因是不完全理解的。这在计算亮度时有影响;当试图?...

Watch this Scientific Journal Video about Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software a

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

本协议描述了一种无标定的方法, 用于在荧光涨落光谱的基础上, 利用商用光扫描显微镜定量测定蛋白质的体外齐聚。给出了正确的采集设置和分析方法。

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

calibration-free-vitro-quantification-protein-homo-oligomerization

Research

JoVE Journal

Biochemistry

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

7.2K 次观看.  University of Oxford. 本协议描述了一种无标定的方法, 用于在荧光涨落光谱的基础上, 利用商用光扫描显微镜定量测定蛋白质的体外齐聚。给出了正确的采集设置和分析方法。

视频: Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

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

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

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

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

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 ...

科研

生物化学

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

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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 ...

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

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample 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 ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

使用生物物理测定表征 PROTAC 中三元复合物的形成

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

使用微流体扩展质量亮度分析浓度范围

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

作者聚焦：使用 MassFluidix 技术表征溶液中低亲和力蛋白质相互作用

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

摘要

探索更多视频

此视频中的章节

原位近接结扎法检测蛋白异构体的 Heterodimerization

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

单分子水平标记均匀性的全蛋白质定量

使用秀丽隐杆线虫中的自动包涵体计数监测体内蛋白质聚集动力学

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成

通过将微流体与质量光度法相结合来分析微摩尔浓度下的蛋白质复合物形成