Name: how to quantify the fraction of photoactivated fluorescent proteins in bulk and in live cells
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells at JoVE.com

我们要感谢斯坦福大学医学院的多罗实验室和神经科学成像服务机构为这一项目提供了设备和空间。

1. 质粒结构
<ol><li>生成双色融合探头。使用哺乳动物细胞表达载体 (见材料表), 其中 mCherry1和pa-mcherry113 与限制位点 agei 和 bsrgi 一起插入. 
</li>
	<li>订购定制寡核苷酸, 以放大含有 a206k 突变的 egfp 和 pa-egfp 的单体变异,即megfp 和 pa-magfp14 , 而没有停止密码子作为sali-bamhi片段。使用 n 端引物 5 '-aat tag tcg acg a...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+mechanism+of+PAmCherry+based+on+crystal+structures+of+the+protein+in+the+dark+and+fluorescent+states.">Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21097-21102 (2009).</li><li>Wiedenmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (45), 15905-15910 (2004).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296 (5569), 913-916 (2002).</li><li>Slocum, J. D., Webb, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Double+Decarboxylation+in+Superfolder+Green+Fluorescent+Protein+Leads+to+High+Contrast+Photoactivation.">A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation.</a> Journal of Physical Chemistry Letters. 8 (13), 2862-2868 (2017).</li><li>Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., Verkhusha, V. 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到目前为止, 还没有方法来批量测定在光活化为荧光的活细胞中表达的 pa-fp 的分数。该协议可用于任何光谱不同的荧光蛋白对。虽然这里的例子是不可逆转的 pa-fp pa-gfp 和 pa-cherry, 但这种方法原则上也适用于光可转换蛋白。然而, 必须仔细选择光谱不同的荧光蛋白, 以最大限度地减少光谱重叠, 因为光可转换荧光蛋白会将其吸收光谱和发射光谱从绿色转移到红色荧光。
如...

2002年, 介绍了第一种广泛适用的光致可激活 (pa-gfp1) 和光可转换 (kaede2) 荧光蛋白。这些光学荧光笔荧光蛋白在用 uv 光照射时改变光谱特性,即它们变得明亮 (可发光荧光蛋白,即pa-fp), 或改变其颜色 (可转换光(fp)。到目前为止, 已经开发出了一些可逆和不可逆的光活性和可光可维化荧光蛋白。在集成或体积研究中, 光学荧光笔被用来研究整个细胞或蛋白质的动态, 以及亚细胞隔间的连接性。此外, 光学荧光笔支持基于单分子的超分辨率成像技术, 如 palm5和 fpalm6.
虽然在光活化或转换过程中的光化学过程已经描述了许多光学荧光笔,甚至在光电激活/转换之前和之后的晶体结构已经提供了7,8、光激活和转换的底层照片物理机制尚不完全清楚。此外, 到目前为止, 只有粗略的估计存在光活化和转换的效率, 即实际表示的光转换或光活化为荧光的荧光蛋白的分数。体外整合研究已报告定量的变化, 在一个凝胶9,10,11的吸收光谱和原生和活化蛋白的数量。
在这里, 我们提出了一个协议, 涉及荧光蛋白嵌合体, 以评估光激活荧光蛋白在体积和活细胞中的分数。每当使用基因编码的荧光蛋白时, 所表达的蛋白质的绝对数量因细胞而异, 是未知的。如果一个表示 pa-fp 的单元在光激活后显示出比另一个单元更明亮的信号, 则如果这种更明亮的信号是由于 pa-fp 的更高表达或 pa-fp 的更有效的光激活而导致的, 则无法区分该信号。为了规范细胞中的表达水平, 我们引入了基因耦合光谱不同荧光蛋白的内部规则。通过将光可激活荧光蛋白的遗传信息与光谱上的始终在线荧光蛋白耦合, 生成了仍可表示为未知总量的内部标尺, 但其相对数量固定在一个固定的和已知的相对数量中。1. 这一战略允许对不同的紫外光光活化方案进行定量表征,即评估可以用不同光活化模式进行光活化的 pa-fp 的相对数量, 从而允许定义比其他方案更有效的光激活方案。此外, 这一战略原则上允许对光激活的 pa-fp 分数的绝对量化进行评估。为此, 重要的是要认识到, 所提出的集成研究是基于强度的, 这使得本协议所规定的分析更加复杂。在比较不同荧光蛋白的荧光强度时, 需要考虑确定测量荧光强度的参数,即不同的分子亮度、吸收率和发射光谱以及 fret 效应。
以比例强度为基础的光活化效率定量为例, 说明了在活细胞中的 pa-gfp 和 pa-cherry, 但原则上广泛适用, 可用于任何光活化荧光蛋白。实验条件。

<table>
	<tbody>
		<tr>
			<td>pEGFP-N1 mammalian cell expression vector</td>
			<td>Clontech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM w/o phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin w/o phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</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>Fugene 6</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
	</tbody>
</table>

how to quantify the fraction of photoactivated fluorescent proteins in bulk and in live cells

荧光和可转换荧光蛋白 (pa-fp) 已被用于荧光活细胞显微镜分析细胞和蛋白质组合的动力学。到目前为止, 还没有方法可以在体积和活细胞中量化有多少 pa-fp 表示的光活化荧光。
在这里, 我们提出了一个涉及内部标尺的协议,即基因耦合光谱分辨 (可光能) 荧光蛋白, 以物理方式量化在被打开的细胞中表达的所有 pa-fp 的分数。荧光。利用该协议, 我们证明了不同的光活化模式产生了不同的光活化效率。利用共聚焦激光扫描显微镜 (clsm) 进行短的大功率光激活, 其光活化效率比 clsm 应用的数百次低电平曝光或宽场照明应用的短脉冲低四倍。虽然该协议已在这里为 (pa-) gfp 和 (pa-) cherry 提供了实例, 但它原则上可以应用于任何光谱上不同的可激活或光可转换荧光蛋白对和任何实验设置。

这里介绍的协议显示了光激活为荧光的荧光蛋白分数的比例定量 (图 1)。此分数因光激活模式的不同而不同。
图 2c显示了利用共聚焦激光扫描显微镜 (clsm) 进行短时间大功率光激活的典型结果。通过像素停留时间和 atof 传输对物镜上测量的激光功率进行滴定后, pa-gfp 的最大光激活效?...

Watch this Scientific Journal Video about How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells at JoVE.com

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

如何定量光活化荧光蛋白在大容量和活细胞中的分数

在这里, 我们提出了一个协议, 涉及基因耦合光谱不同的光可激活和荧光蛋白。这些荧光蛋白嵌合体允许定量的 pa-fp 分数是光活化的荧光,即光活化效率。该协议揭示了不同的光活化模式产生不同的光活化效率。

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

我们在这里介绍一个协议，涉及基因耦合光谱上独特的光可操作和荧光蛋白。这些内部标尺允许量化被照片激活为荧光的PAFP分数。到目前为止，还没有一种方法可以批量量化生命细胞中有多少PAFP表达的PAFP被光激活到荧光。PA-GFP和PA Cherry在活细胞中提出了光活化效率的量化，但原则上广泛适用，可用于任何实验条件下的任何光能荧光蛋白。每当使用基因编码的荧光蛋白时，表达的蛋白质的绝对数量因细胞而异，而且未知。为了标准化细胞之间的表达，我们引入了基因耦合光谱上不同的荧光蛋白的内部标尺。通过将光能度荧光蛋白的遗传信息与荧光蛋白上始终具有光谱差异的遗传信息，产生的光度蛋白内部标尺仍以未知总量表示，但以一比一的固定已知相对量表示。开始构造质粒，如随附的文本协议中所述。此外，培养在标准细胞系的透视介质使用酚红色自由版本。当准备开始实验时，使用不带酚红色的三辛来收获细胞，以减少背景花期。收获后，用生长在T 12.5细胞培养瓶中的汇合细胞培养的细胞悬浮液填充2ml移液器。将一滴加入八室玻璃滑梯的每个腔室。在标准细胞培养条件下孵育滑梯24小时。接下来，使用商业试剂按照分配器协议转染细胞。使用 Gfp 樱桃、PA-GFP 樱桃和 Gfppa 樱桃嵌合子转换细胞。然后将细胞返回培养箱，以允许蛋白质表达、折叠和成熟。转染后总共20小时后，将细胞转移到装有加湿和加热环境室预加热至37的共合体荧光显微镜的阶段

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

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

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 ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...