Name: assessing protein interactions in live-cells with fret-sensitized emission
Uploaded: 2021-04-22T14:00:00
Description: Watch this Scientific Journal Video about Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission at JoVE.com

我们要感谢斯坦福大学医学院的神经科学成像服务为这个项目提供设备和空间。这项研究得到了斯坦福癌症研究所和斯坦福妇科肿瘤科的校内资助，以及匈牙利国家研究、发展和创新办公室的GINOP-2.3.2-15-2016-00026、GINOP-2.3.3-15-2016-00030、NN129371、ANN135107。

1. 质粒构建
<ol><li>为了生成eGFP-mCherry1融合探针，使用N1哺乳动物细胞表达载体（参见材料表），使用限制性位点AgeI和BsrGI插入mCherry110。</li>
	<li>使用以下寡核苷酸在没有终止密码子的情况下扩增 eGFP11 作为 SalI-BamHI 片段：N 末端引物 5'-AAT TAA CAG TCG ACG ATG GTG AGC AAG GGC GAG G 3' 和 C 末端引物 5'-AAT ATA TGG ATC CCG CTT GTA CAG CTC GTC CAT GC 3'。<...

<ol>
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所提出的协议详细介绍了使用基因偶联的一对一荧光蛋白校准探针来量化FRET使用共聚焦显微镜检测受体的敏化发射和供体分子的淬灭。该方法可用于评估不同亚细胞区室中活细胞生理环境中的蛋白质相互作用。通过应用所提出的算法来计算图像每个像素的FRET效率（逐像素FRET），可以进一步提高空间分辨率。基于强度的绝对FRET效率测定需要确定串扰，此处用 S 因子量化，以及通过给定的显...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated from:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine
to:
Gy&#246;rgy V&#225;mosi1 
Sarah Miller2 
Molika Sinha2 
Maria Kristha Fernandez2 
Gabor Mocs&#225;r1 
Malte Renz2 
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen 
2Gynecologic Oncology Division, Stanford University School of Medicine

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62241">Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission</a>. The Authors section was updated.

Erratum: Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

基于显微镜的福斯特共振能量转移（FRET）分析允许评估活细胞中蛋白质之间的相互作用。它提供空间和时间信息，包括有关细胞中发生相互作用的位置和亚细胞区室中发生以及这种相互作用是否随时间变化的信息。
Theodor Förster于1948年奠定了FRET的理论基础1.FRET是从激发的供体到受体分子的能量的无辐射转移，取决于分子的距离及其过渡偶极子的相对方向以及供体发射和受体吸收光谱之间的重叠。能量转移速率与供体-受体距离的六次方成反比。因此，FRET可用于测量1-10nm范围内的分子接近度。
FRET与供体分子的其他去激发过程竞争，并导致受体的所谓供体淬灭和敏化发射。供体猝灭是发射供体光子数量的减少，而敏化发射是发射受体光子的增加。许多显微FRET分析使用荧光强度测量，包括受体光漂白2，供体光漂白2或受体3的FRET敏化光漂白。
在这里，提出了一种分步实验方案和数学算法，以使用供体淬灭和受体敏化发射4，5（通常称为比率FRET）来量化FRET的方法。关于如何近似敏化发射的许多协议已经发布，很少有量化绝对FRET效率6，7，8，9。活细胞中FRET效率的定量需要确定（i）荧光蛋白的串扰（光谱溢出或渗漏），以及（ii）显微镜装置的检测效率。虽然串扰可以通过仅表达一种荧光团的细胞成像来评估，但对供体和受体荧光的相对检测效率的评估更为复杂。它至少需要了解产生测量信号的供体和受体分子数量的比率。然而，活细胞中表达的荧光团数量因细胞而异，并且是未知的。所谓的α因子表征来自单个激发供体和受体分子的相对信号强度。了解该因子是在具有可变受体-供体分子比例的样品中进行定量比例法FRET测量的先决条件，就像在使用荧光蛋白进行活细胞成像时遇到的一样。使用1对1供体-受体融合蛋白作为校准探针可以确定α因子，也可以作为阳性对照。这种基因偶联探针由细胞以未知的总量表达，但以固定且已知的相对量一对一表达。以下协议列出了如何构建1对1探头以及如何使用它来量化FRET效率。在增刊中可以找到包含所有公式的电子表格，读者可以使用该电子表格在相应的列中输入自己的测量值，如下所述。
虽然该协议使用GFP-Cherry供体/受体对，但所提出的方法可以用任何其他FRET对执行。 补充文件 1 提供了有关青黄色对的详细信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA without phenol red (10x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Clontech mCherry N1 vector</td>
			<td>Addgene</td>
			<td>3553</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM without phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene 6</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>LabTek 8-well chambers #1.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565470</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing protein interactions in live-cells with fret-sensitized emission

福斯特共振能量转移（FRET）是从激发的供体到受体分子的能量的无辐射转移，取决于分子的距离和方向以及供体发射和受体吸收光谱之间的重叠程度。FRET允许研究活细胞中蛋白质随时间变化和不同亚细胞区室中的相互作用。文献中已经描述了使用显微镜测量FRET的不同基于强度的算法。在这里，提供了一种协议和算法，用于基于测量受体的敏化发射和供体分子的淬灭来量化FRET效率。活细胞中比例FRET的定量不仅需要确定荧光蛋白的串扰（光谱溢出或渗漏），还需要确定显微镜装置的检测效率。此处提供的协议详细说明了如何评估这些关键参数。

图1分别显示了在供体通道通道1（488，505-530 nm），转移通道通道2（488，>585 nm）和受体通道3（561，>585 nm）中获得的图像。仅表达GFP，仅表达樱桃，共表达GFP和樱桃以及表达GFP-樱桃融合蛋白的细胞的代表性图像。绘制在表达GFP-樱桃融合蛋白的NRK细胞（阳性对照，图2A）和共表达GFP-樱桃（阴性对照，图2B）的NRK</str...

Watch this Scientific Journal Video about Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission at JoVE.com

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

两个荧光团分子之间的福斯特共振能量转移（FRET）可用于研究活细胞中的蛋白质相互作用。在这里，提供了有关如何通过使用共聚焦激光扫描显微镜检测受体的敏化发射和供体分子的淬灭来测量活细胞中的FRET的协议。

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

该协议描述了使用供体淬灭和受体敏化发射量化FRET的分步实验过程和数学算法。活细胞中FRET效率的定量需要确定荧光蛋白的串扰以及显微镜装置中荧光团的相对发射和检测效率。串扰可以通过仅表达一个荧光团的细胞成像来评估。评估供体或受体分子的检测效率需要了解产生测量信号的供体和受体分子的数量之比。活细胞中表达的荧光团数量因细胞而异，并且是未知的。该方法中使用的校准探针是一对一的供体-受体融合蛋白，可以确定供体和受体分子的相对检测效率。首先，使用带有mCherry 1的N1哺乳动物细胞表达载体生成EGFP mCherry 1融合探针。设计寡核苷酸以扩增没有终止密码子的EGFP，作为细胞一个BAM h1片段。绿色和红色荧光蛋白之间的五个氨基酸接头应产生GFP樱桃供体 - 受体对的平均FRET缺乏0.25至0.3。使用不含酚红的任何细胞系和培养基来减少背景荧光。一旦细胞汇合80%，用一毫升0.05%胰蛋白酶EDTA分离它们，并通过从5毫升细胞悬液中加入三滴，将约10， 000个细胞接种到八孔板的每个孔中。接种24小时后，使用适当的转染培养基转染细胞。在活细胞成像前孵育细胞20小时，以允许适当的荧光蛋白表达，折叠和成熟。用共聚焦扫描显微镜在37摄氏度的加湿和加热的环境室中对细胞进行成像。为了在生理pH下缓冲细胞培养基，添加20毫摩尔HEPES以使细胞培养基二氧化碳独立。然后将载玻片安装在显微镜上。使用氩离子激光器的488纳米线激发GFP，使用561纳米二极管激光器激发樱桃。设置 488 纳米激光器，用于在通道一中通过 505 至 530 纳米的发射带进行激发，在通道二中通过超过 585 纳米的长通滤光片进行激发。使用 561 纳米激光器在通道三中使用超过 585 纳米的长通滤光片进行激发。依次激发两个激光器，并将成像模式设置为在每行之后切换，以便 512 x 512 像素图像的激发在每行之后交替。设置一个由三个图像组成的迷你时间序列，以检测是否发生明显的光漂白，并在必要时降低激光功率。首先，对表达GFP樱桃融合构建体的细胞进行成像。设置定义共聚焦图像中每个像素的时间积分激光强度的参数。使用63倍油镜并将变焦设置为3倍，以足够的放大倍率和分辨率对整个细胞进行成像。瞄准 70 到 80 纳米的像素大小。将像素停留时间设置为 2 到 4 微秒，并将 488 纳米和 561 纳米激光器的 AOTF 传输设置为图像，以便图像显示出良好的信噪比，没有任何漂白，也没有指示荧光强度饱和的像素。调整 488 和 561 的激光功率，使通道 1 和通道 3 中的信号电平相似。将照片倍增增益设置为 600 到 800。图15至20表达GFP樱桃融合蛋白的细胞，表达GFP，樱桃，GFP和樱桃的细胞以及非横断面细胞。然后对细胞进行成像，分别共表达与GFP和樱桃偶联的目标蛋白。要计算FRET，请测量通道1中的供体信号，该供体通道具有488纳米激发，发射带为505至530纳米。然后测量通道三中的受体信号，该受体通道用561纳米激发并在585纳米以上发射。最后，测量通道二中的FRET信号，该传输通道具有488纳米的激励和585纳米以上的发射。通道二中的信号是四个不同分量的总和：从淬灭供体信号到具有串扰因子S1的超过585个检测通道的光谱溢出，来自具有串扰因子S2的488纳米光直接激发的受体信号，FRET从激发的供体分子中敏化发射的受体， 和背景信号。在供体通道、转移通道和受体通道中获得图像，显示仅表达GFP，仅樱桃，共表达GFP和樱桃的细胞以及GFP樱桃融合蛋白。绘制了表达GFP樱桃融合蛋白和共表达GFP樱桃的NRK细胞中计算的平均细胞FRET效率与每个细胞中的受体-供体比强度比或分子比的关系图。此处显示了表达GFP樱桃融合蛋白，共表达GFP和樱桃作为阴性对照以及表达Ashwell-Morell受体受体亚基的细胞的标准化逐像素FRET图像。大鼠肝凝集素的RHL1和2在质膜的细胞质侧用GFP和樱桃标记。绘制表达GFP RHL1和樱桃RHL2以及GFP RHL1δ原液和樱桃RHL2的细胞的平均细胞FRET效率与受体-供体分子比的关系图。所提出的定量FRET方法允许以下内容。检测活细胞生理环境中的蛋白质相互作用。蛋白质相互作用随时间的变化。亚细胞区室中相互作用的差异低至共聚焦图像的逐像素水平。以及检测到的FRET信号对活细胞中表达的分子受体与供体比的依赖性。

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

4D Imaging of Protein Aggregation in Live Cells

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

The MultiBac Protein Complex Production Platform at the EMBL

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...