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 (如图 2a所示)。使用自动 detrend 算法允许精确的亮度计算。
在获取图像时遵循一些规则是很重要的。首先, 在共焦体积中分子的停留时间, 从而确定其扩散常数, 以设定共焦系统的正确参数;标记分子的停留时间需要驻留在像素停留时间和帧时间7之间。选择正确的荧光是至关重要的。一个明亮和稳定的荧光是必要的良好的信号噪声和最小的漂白。激光功率应保持相对较低以避免漂白。光子计数1或2每像素是充足的。保持低计数对于避免检测器堆积也是至关重要的。该技术处理低光子预算比它处理漂白。在本议定书中使用了 mVenus;这是一个相对明亮的荧光, 相当于 eGFP19。另一种选择是使用 nanoboosters, 选择性地瞄准荧光蛋白;这些比传统的显影20还要明亮得多。
在硬件方面, 数字光子计数探测器使量化更容易, 但 N & B 是可能的模拟探测器, 只要一个已经恢复了一些采集参数。8
尽管像各向异性的其他方法可以检测到二聚化, 随着新软件的问世, 简化分析, N & B 单独作为一种可访问的方法来检测和量化蛋白质相互作用和齐聚, 无论是在体外和活细胞中。

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 border="0" cellpadding="0" cellspacing="0" style="width:997px;" width="997">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">RosettaTM (DE3) pLysS cells</td>
			<td style="width:123px;">Novagen</td>
			<td style="width:344px;">70956-3</td>
			<td style="width:200px;"></td>
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			<td height="40" style="height:40px;">Ampicillin</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">PubChem Substance ID 329824407</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chloramphenicol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>PubChem Substance ID: 24892250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LB starter culture</td>
			<td style="width:123px;">QIAGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LB medium</td>
			<td style="width:123px;">QIAGEN</td>
			<td height="40" style="height:40px;width:344px;&quot;">https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IPTG</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">PubChem Substance ID 329815691</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IMAC buffer</td>
			<td>Medicago</td>
			<td>09-1010-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA-free protease inhibitors&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>11873580001</td>
			<td></td>
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			<td height="40" style="height:40px;">TALON resin</td>
			<td>Clonetech</td>
			<td></td>
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			<td height="40" style="height:40px;">Nickel sepharose</td>
			<td style="width:123px;">GE Healthcare</td>
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			<td height="40" style="height:40px;">S200 16/60 column</td>
			<td style="width:123px;">GE Healthcare</td>
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			<td>Ibidi</td>
			<td>80827</td>
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</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;当试图确定单体-二聚体转变时, 这方面是至关重要的。通过将自动 detrend 算法应用于图 2a所示的数据, 对强度跟踪进行了正确的修正, 为解决方案中 FKBP12F36V-mVenus 的亮度提供了更准确的值。在加法之前 BB, 没有 detrending, b = 1.026, 而在 detrending 以后, b = 1.005 (图 2B)。
FKBP12 的测定F36V-mVenus 单体-二聚体过渡体外
在 100 nM 溶液中, 获得了2万种纯化 FKBP12F36V-mVenus 的图像序列, 如协议部分所述。在获得1万帧后, homodimerizer 药物 BB 被添加到溶液中。分析了每个连续的5000帧序列 (图 3)。平均亮度5分钟后 BB 加法是B = 1.010, 这是一个2倍的增长, 表明 FKBP12F36V 二聚体。过程的动力学如图 3b所示;BB 加到全 FKBP12F36V 二聚化之间的延迟约为2分钟。
蛋白质团聚体的鉴定
在有限数量的帧和强度跟踪中检测到许多蛋白质聚集物 (图 4)。这些聚合在溶液中自然发生, 当使用蛋白质时, 可以通过 sonicating 和/或增加蛋白质稀释来消除。然而, 在一些生物学问题中, 人们需要检测单体和大团聚体之间的过渡。图 4显示了一个例子, FKBP12F36V 蛋白聚合体通过观察量扩散 (图像 16 x 16);对于我们以前的计算, 这些数据被丢弃了, 但是,图 4中显示了一个示例, 表明可以使用相同的设置和方法检测和描述这些聚合。乍一看, 当评估5000图像, 你可以恢复平均亮度为 FKBP12F36V-mVenus 治疗的 BB (B = 1.010)。查看原始亮度图像, 可以清楚地看到, 虽然, 一个感兴趣的区域约8像素的高值B ≈1.080。这个感兴趣的区域在 t = 34 三十七年代的几个框架重合, 在那里 FKBP12F36V 聚集在观察区域附近扩散。骨料在观测量中没有保持足够长的时间, 以准确确定其大小。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58157/58157fig1.jpg"> 图 1.应用 N & B 检测蛋白质单体-二聚体在溶液中的转变。(a)激光扫描显微镜 (LSM) 的简化光学路径, 配备激光源 (在 mVenus 标记的蛋白质的情况下设置为 514 nm) 定向 (蓝色箭头) 朝向浸入目标 (在我们的例子中是63X1.4NA 油), 照亮了 100 nm 溶液的FKBP12F36V-mVenus 解决方案。发射荧光 (绿色箭头) 通过一个分色镜, 并定向到一个带通滤波器, 清除发射光, 和一个针孔设置在1通风单元位于右侧的点数字探测器能够光子计数。(b)通过 16 x 16 像素扫描共聚焦的光照量, 以照亮单个 FKBP12F36V-mVenus 分子, 进入并退出高斯形状共焦体积, 产生一系列荧光强度波动。(c)随时间推移而获得的图像系列。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58157/58157fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58157/58157fig2.jpg"> 图 2.为了准确测量溶液中单体的数量, 需要自动 detrending。(a)按照《议定书》一节所述, 获得了一叠 5000 16 x 16 像素图像。第一帧的强度与平均时间分辨强度剖面一起显示, 这表明可能与漂白和其他溶剂和/或 photophysic 效应有关的长期波动。不管这些长期波动的原因是什么, 它们会影响亮度计算, 因此需要 detrending。没有自动 detrend, 你得到B = 1.026, 而在自动 detrending 之后, b = 1.005。此外, 亮度没有 (左面板, 第二行) 和 (右面板, 第二行) 平滑过滤显示。( b)在 (a) 中提出的相同数据是去趋势的, 并显示了强度和亮度方面的结果。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58157/58157fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58157/58157fig3.jpg"> 图 3.体外 N & B 能实时解决 FKBP12F36V 单体与脂肪酸之间的过渡。dimerizer 药物 BB 的加法导致了真实的亮度的二倍的增量在2分钟以后 (从0.005 到0.010 在真正的分子亮度)。(a)第一帧的强度与去趋势图像的平均强度剖面一起显示。亮度没有 (左面板, 第二行) 和与 (右面板, 第二行) 平滑过滤显示。(b)平均亮度 (每5000帧) 使用真正的分子亮度ε绘制。二聚化发生2分钟后 BB 加法 (t = 1min) 在t = 4 分钟。拟合曲线是在 dimerizer 药物 (BB) 添加后, 应激二聚化的 sigmoidal 函数。误差线表示每个时间点的亮度分布的标准错误。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58157/58157fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58157/58157fig4.jpg"> 图 4.体外 & B, 以解决溶液中蛋白质集料的大小。(a)用于获取 FKBP12F36V-mVenus 的时间分辨强度去趋势跟踪与亮度图像 (第二行) 一起显示。(b)时间分辨的去趋势强度跟踪显示的强度最高, 以红色虚线圆圈 (左上板) 突出显示。显示与此特定时间 (t = 34 秒) 对应的第一个强度帧, 红色箭头显示进入光照区域的 FKBP12F36V-mVenus 聚合。仔细研究平滑的亮度图像显示了一个感兴趣的区域, 其中包含高寡聚状态, 其余的图像包含单体和脂肪酸之间的混合, 平均为B = 1.008。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58157/58157fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.

This method provides a gravimetric quantification of humic substances (e.g., humic and fulvic acids) on an ash-free basis, in dry and liquid materials from soft coals (i.e., oxidized and non-oxidized lignite and sub-bituminous coal), humate ores and shales, peats, composts and commercial fertilizers and soil amendments.

腐植酸盐矿石、DOC、腐殖化材料和含腐殖质的商业产品中腐殖酸和黄腐酸的定量

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and ...

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (&#9642;OH) to irreversibly label a protein&#8217;s solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of &#9642;OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein studies.

无激光羟基基蛋白足迹，对蛋白质进行更高级的结构分析

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...