Name: in vivo quantification of g protein coupled receptor interactions using spectrally resolved two-photon microscopy
Uploaded: 2011-01-19T17:30:00
Description: Watch this Scientific Journal Video about In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy at JoVE.com

这项工作是由威斯康星大学密尔沃基分校的研究增长倡议“，威斯康星州的生物医学和卫生技术学院，和布拉德利基金会的支持。

1。质粒设计感兴趣的蛋白质融合两种不同的荧光标记，如下所述。荧光标记不仅提供有关细胞内的蛋白质的位置，而且还蛋白 1的同源齐聚定量信息的信息。荧光共振能量转移（FRET）2-4技术是用来积累有关的蛋白质相互作用5-10的信息。 FRET技术利用能量转移的原则，可以从光激发态分子（通常称为捐助，D）会出现一个平淡的分子（通常称为受体，一?...

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在此演示中，我们说明了如何确定的规模和在体内的蛋白质，低聚物复杂的结构信息。虽然所给出的数据是从一个特定的膜受体在酵母细胞中表达（即Ste2p），方法是包罗万象，它可应用于任何类型的蛋白质在任何类型的细胞，只规定表示，这些蛋白质与相应的荧光标记物标记。对本协议的未来修改将涉及加强文书，以达到更高的扫描速度，企图监视时间依赖性蛋白低聚物的规模和结构的?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Peptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1420</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1422</td>
<td>&nbsp;</td>
<tr>
<td>Polyethlyene Glycol 4000</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR2-605</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Nitrogen Base w/o (NH4)2SO4 and amino acids</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>DF0335-15-9</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Synthetic Drop-out Medium Supplements</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>Y2001</td>
<td>&nbsp;</td>
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<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D16-1</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S70210</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Sulfate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A702-500</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>424090010</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP385</td>
<td>&nbsp;</td>
<tr>
<td>Histidine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP382</td>
<td>&nbsp;</td>
<tr>
<td>Plan Achromat Infinity Corrected 100x Oil Immersion Objective NA=1.43</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spectrally resolved two photon microscope</td>
<td>&nbsp;</td>
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in vivo quantification of g protein coupled receptor interactions using spectrally resolved two-photon microscopy

在活细胞内蛋白质相互作用的研究是一个重要的研究领域，因为这些信息积累了双方利益的工业应用，以及增加基本的生物知识。福斯特（荧光）共振能量转移（FRET）技术已被捐助者之间的电子激发态的分子和附近的受体分子经常利用在活细胞内蛋白质相互作用的研究。感兴趣的蛋白质，是两个不同类型的荧光探针标记，并在生物细胞中表达。然后兴奋的荧光探针，通常用激光，和排放所产生的荧光探针的荧光光谱特性被收集和分析。有关蛋白质相互作用的程度信息嵌入在光谱的排放数据。通常情况下，细胞必须被扫描的次数，以积累足够的光谱信息准确量化地区的每一个细胞内的利益的蛋白质相互作用的程度。然而，这些地区的分子组成，在收购过程中可能会发生变化，限制了明显的FRET效率的定量值平均在整个细胞空间的决心。通过一个光谱分辨的双光子显微镜，我们能够获得利益的样本只有一个完整的激励扫描后的光谱解析影像的全套。从这个像素级的光谱数据，一个地图FRET整个细胞的效率计算。通过运用一个简单的理论低聚复合物的FRET实验获得的分配的FRET效率在整个细胞，单次扫描光谱解决，揭示正在研究有关的计量和结构低聚物复杂的信息。在这里，我们描述生物细胞 （酿酒酵母）表达两个不同类型的荧光探针标记的膜受体（无菌2α-因子受体）的准备过程。此外，我们说明了使用荧光光谱解决双光子显微成像系统数据收集所涉及的关键因素。使用此协议的可扩展研究可以在与它连接到一个荧光标记的活细胞中表达的任何类型的蛋白质。

Watch this Scientific Journal Video about In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy at JoVE.com

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

通过采用一个光谱分辨的双光子显微成像系统，像素级别的地图福斯特共振能量转移（FRET）效率得到表达假设形成同源寡聚体复合物的膜受体细胞。从FRET的效率图，我们正在研究能够估计约低聚物复杂的化学计量信息。

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Research

JoVE Journal

Biology

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

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

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, ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...