Name: a colorimetric assay of citrate synthase activity in drosophila melanogaster
Uploaded: 2020-01-16T12:00:00
Description: Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

这项工作得到了国家自然科学基金（31401013和31471010）、上海市科委、上海浦江计划（14PJ1405900）和自然科学基金的资助。上海 （19ZR1446400）。

1. 色度色酸盐合成酶活性测定法D. 黑色素酶 16
<ol><li>收集10只成年苍蝇，每个样本。收集每个基因型的至少三联体样本。</li>
	<li>在每个样品的1.5 mL试管中制备500 μL的冷萃取缓冲液，含有20 mM HEPES（pH = 7.2）、1 mM EDTA和0.1%三吨X-100。</li>
	<li>在麻醉垫上用CO2麻醉成年苍蝇，并分离所需的组织。例如，要分离成年苍蝇胸斧，用一对钳子固定苍蝇胸斧，然后用剪刀沿胸?...

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以果蝇为模型的代谢研究必须考虑到苍蝇的遗传背景、饮食和种群维持。为了避免不同遗传背景对锡酸盐合成酶活性的测量的影响，不同的果蝇菌株应回到10代的对照菌株。实验中使用的所有果蝇菌株的遗传背景是w1118，所以我们用w1118作为对照。一般来说，我们认为w1118是一个很好的控制，因为它是大多数果蝇<...

线粒体在大多数真核生物中最出名的是产生能量的细胞器，它通过三甲苯酸循环（即克雷布斯循环）和氧化磷酸化产生能量货币ATP。线粒体在许多其他生理过程中也起着重要作用，如细胞凋亡1，Ca2+平衡2，3，反应性氧化物种（ROS）第4代，和内质神经素（ER）-应激反应5。线粒体功能障碍可以影响身体的任何器官在任何年龄，是代谢，老化相关6，和神经退行性疾病7的主要原因。完整的线粒体与线粒体功能有机械关系。因此，正确定量完整的线粒体质量对于评价线粒体功能8非常重要。Citrate合成酶是三聚体酸循环9第一步的速率限制酶，在真核细胞内的线粒体基质中局部化，因此可用作完整线粒体质量9、10存在的定量标记。锡酸盐合成酶活性也可用作完整线粒体蛋白11、12的正常化因子。
果蝇，果蝇，果蝇黑色素，是研究线粒体功能的一个很好的模型系统，因为许多分子和途径，在线粒体中起着关键作用，从果蝇进化保存到人类13，14，15。在这里，我们提出了一种快速而简单的方法，通过果蝇组织中的色度测定以96孔板格式对质16进行色度测定，以快速而简单的方法测量锡酸盐合成酶活性。在锡酸盐合成酶活性测定中，果蝇组织中的锡酸合成酶均质化酶催化草酸与乙酰辅酶A（乙酰CoA）的反应，形成锡酸盐CoA-SH和H+。CoA-SH 随后与 5，5'-dithiobis-（2-硝基苯甲酸） （DTNB） 发生反应，以生成彩色产品，2-硝基-5 硫苯甲酸酯 （TNB），可在 412 nm 处轻松测量分光光度。色酸盐合成酶活性可以通过颜色生产速率来反映。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>V900477</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base)</td>
			<td>Sigma-Aldrich</td>
			<td>V900483</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Sigma-Aldrich</td>
			<td>A2181</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithio-bis-nitrobenzoic acid (DTNB)</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>V900106</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaloacetate</td>
			<td>Sigma-Aldrich</td>
			<td>O4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle</td>
			<td>Sangon</td>
			<td>F619072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle motor</td>
			<td>Tiangen</td>
			<td>OSE-Y10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>Eon</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein BCA Assay kit</td>
			<td>Beyotime</td>
			<td>P0010S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>WPI</td>
			<td>14124</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon</td>
			<td>A110694-0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay of citrate synthase activity in drosophila melanogaster

线粒体在细胞代谢中起着最突出的作用，通过氧化磷酸化产生ATP并调节各种生理过程。线粒体功能障碍是许多代谢和神经退行性疾病的主要原因。完整的线粒体对其正常功能至关重要。酶Citrate合成酶在线粒体基质中局部化，因此可用作完整线粒体质量的定量酶标记。鉴于许多在线粒体中具有重要功能的分子和途径在人类和果蝇之间高度保存，并且果蝇体内有一系列强大的遗传工具，果蝇是研究线粒体功能的良好模型系统。在这里，我们提出了一个协议，用于快速和简单地测量在组织同质体中与成苍蝇的胆酸合成酶活性，而无需隔离线粒体。该协议也适用于测量幼虫、培养细胞和哺乳动物组织中的锡酸盐合成酶活性。

图1给出了一个使用锡酸盐合成酶活性色测定测量不同基因型的果蝇胸肌组织均质的在412nm处获得OD吸收率的动力学曲线示例。众所周知，PGC-1+是线粒体生物成因的主要调节器。PGC-1+在果蝇和人类之间具有功能保护性。德罗索菲拉RNF34 （dRNF34） 是一种E3泛素连带酶，用于果蝇PGC-1+，dPGC-1，促进dPGC-1蛋白降解17...

Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

我们提出了一种对胆酸合成酶活性的色度测定方案，用于定量果蝇组织均质中完整的线粒体质量。

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

果蝇是研究线粒体功能的好模型系统。在这里，我们提出了一个协议，用于测量成人苍蝇组织中柠檬酸盐合成酶活性。该协议不需要线粒体隔离。它快速而简单，适合测量幼虫、培养细胞和哺乳动物组织中柠檬酸盐合成酶活性。首先收集每个样本的成年苍蝇，确保收集每个基因型至少三分样本。在每个样品的 1.5 毫升试管中制备 500 微升的冰冷萃取缓冲液，内有 20 毫摩尔 HEPES、1 毫摩尔 EDTA 和 0.1%三吨 X-100。麻醉垫上用二氧化碳麻醉成年苍蝇。要隔离胸部，请用一对钳子固定它们，然后用一把剪刀沿着胸部和腹部的边界切割，从而隔离飞腹。收集柠檬酸盐合成酶活性测定的飞胸。立即将10只成年苍蝇的胸被转移到100微升的冰冷萃取缓冲液中，用冰上的害虫将其均匀化。在冰上均质5至10秒，让样品在冰上坐5秒钟，然后重复，直到所有组织完全均质。每个均质样品的10微升均匀度进入一个新的管，用于蛋白质含量测量，使这些管子也保持在冰上。在每个剩余样品中加入 400 微升的冰冷萃取缓冲液，总体积为 500 微升，并上下移液器轻轻混合。对于每次反应，在新鲜准备的反应溶液的150微升中加入一微升稀释细胞解酸。通过轻轻移液彻底混合，确保避免气泡形成，然后使用板读卡器测量吸收度在412纳米每10至30秒在25摄氏度下4分钟。将数据绘制为一段时间的吸收，然后计算曲线线性部分的斜率。最后，将反应速率或斜率除以样品蛋白浓度，使柠檬酸盐合成酶活性正常化。据推测，在果蝇肌肉中敲掉dRNF34将增加线粒体柠檬酸盐合成酶活性，而dPGC-1的击倒将扭转这种增加。为每次测定建立了初始线性酶速率;趋势线的斜率表示最大反应速率，相当于不同基因型的最大柠檬酸盐合成酶活性。最大柠檬酸盐合成酶活性从动力学曲线计算，并由蛋白质浓度进行规范化。结果表明，飞胸的合成酶活性确实随着肌肉特异性dRNF34的击倒而增加，而增加的增强被肌肉特异性dPGC-1击倒逆转。当样品均质时，重要的是对管子进行尖端和检查均质，以确保没有血块，并且管中的组织完全均匀。采集样品后，所有样品处理都应在冰上进行。柠檬酸盐合成酶活性测定用于量化完整的线粒体质量。正确定量完整的线粒体质量对于评价线粒体功能非常重要。

Research

JoVE Journal

Biochemistry

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

A Fluorescence-based Assay of Phospholipid Scramblase Activity

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone (Video) | JoVE

RGBradford：使用智能手机相机进行蛋白质定量

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...