Name: monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret
Uploaded: 2012-01-27T12:00:00
Description: Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

作者支持由瑞典研究委员会，瑞典战略研究，瑞典癌症协会，瑞典的儿童癌症协会，阿克Wibergs基础和Jeanssons基础的基础。

1。探头引入到细胞<ol><li>一个基于FRET的探头和质粒抗共转染的细胞。选择转染的方法，该方法是有效的，在你感兴趣的细胞类型。对于U2OS细胞，标准的磷酸钙转染方法给予足够的结果15。 </li><li>选择适当的抗生素至少7天的细胞。这丰富的数额细胞表达探头和限制有毒的表达水平，表达水平，严重影响细胞周期的细胞量。 </li><li> （可选）选择稳定的克隆。 </li><li>使用固定或活细胞，?...

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监测苦恼整个细胞周期需要评估短期应对外部刺激时，是同样重要的的考虑。首先，细胞周期的进程很容易扰动压力信号，要求保持在最低限度，光毒性。其次，所有的记者可能影响滴定激酶，磷酸或相互作用域的细胞过程。可能是最简单的方法来评估，如果有足够的实验条件来衡量细胞周期的长度从有丝分裂有丝分裂和独立实验的荧光成像探针表达比较。 在监测的关键因?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 目录编号 </td><td> 公司 </td></tr><tr><td>莱博维茨的L - 15，无酚红</td><td> 21083-027 </td><td> GIBCO公司，生命科技</td></tr><tr><td>的DMEM + Glutamax，我</td><td> 31966 </td><td> GIBCO公司，生命科技</td></tr><tr><td>胎牛血清（FBS） </td><td> SV30160.03 </td><td> Hyclone公司</td></tr><tr><td> 0.05％胰蛋白酶EDTA </td><td> SH30236.01 </td><td> Hyclone公司</td></tr><tr><td>青霉素链霉素</td><td> SV30010 </td><td> Hyclone公司</td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO公司，生命科技</td></tr><tr><td>嘌呤霉素</td><td> P8833 </td><td> Sigma - Aldrich公司</td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

福斯特共振能量转移（FRET）为基础的记者1允许在活细胞中的内源性蛋白激酶和磷酸酶活性的评估。这种探头通常由CFP和YFP的变种，由phosphorylatable序列和磷酸结合结构域进行干预。磷酸化后，探头改变构象，CFP和YFP之间的距离或方向的变化，结果，导致在变化FRET效率（图1）。几个探头在过去十年中，已公布的监测多个激酶和磷酸酶的活性的平衡，其中包括，PKD的5 4 3 PKB，PKC，ERK的6，7 JNK 的 CDK 18， 极光B 9 2，PKA的记者和 PLK1 9 。鉴于模块化设计，额外的探头有可能出现在不久的将来10。 通过细胞周期的进展是受压力signali吴途径11。值得注意的是，不同的细胞周期调节在不受干扰的增长，而当细胞恢复应力 12。定时通过细胞周期的细胞成像，因此需要特别小心。这将成为一个问题，尤其是当用人比例成像，因为两个图像具有很高的信噪比，需要正确地解释结果。成比例的FRET成像细胞周期依赖激酶和磷酸酶活性的变化为主被限制在细胞周期 8,9,13,14分节。 在这里，我们讨论的方法来监视基于FRET的探头使用整个人类的细胞周期的比例成像。方法依赖于设备，是在生命科学领域的许多研究人员，并不需要显微镜或图像处理专业知识。

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

基于FRET的记者越来越多地用于监测活细胞中的激酶和磷酸酶的活动。在这里，我们描述了如何使用基于FRET的记者，以评估目标磷酸化细胞周期依赖性变化的方法。

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Research

JoVE Journal

Biology

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

从老鼠血液通过大隐静脉和心脏穿刺“

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

科研

生物学

Analysis of Cell Cycle Position in Mammalian Cells

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

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研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

在活细胞悬浮液荧光测定G蛋白选择性GPCR构象测量使用FRET传感器

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...