Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
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Author:
Date:
2016-08-05
[Abstract] The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro ...
[摘要] 凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用,并且还依赖于线粒体的形状和大小。在过去的研究中使用细胞模型不是理想的调查如何复杂的多因素相互作用调节线粒体外膜透化(MOMP)的分子机制。分离系统已被证明是将MOMP解构成各个步骤并研究每个子集的MOMP调节剂的行为的范例。特别地,分离的线粒体是BCL-2家族蛋白的体外研究的关键,BCL-2家族蛋白是直接控制凋亡的线粒体途径的促存活和促凋亡蛋白的复合家族(Renault > et al 。,2013)。 在这个协议,我们描述三个补充程序用于实时调查使用孤立的线粒体MOMP调节器的影响。第一种方法是"肝线粒体分离",其中从小鼠中分离肝脏以获得线粒体。 "用JC-1和大小分级分离的线粒体标记"是描述标记,按大小分级并标准化线粒体亚群的方法的第二个方法。最后,"实时MOMP测量"协议允许在孤立的线粒体上实时跟踪MOMP。上述程序用于在体外确定线粒体膜形状在分离的细胞和分离的线粒体水平上的作用(Renault等人,2015)。
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Quantitative Analysis of Cellular Diacylglycerol Content
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Author:
Date:
2014-08-05
[Abstract] Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid.
This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to ...
[摘要] 二酰基甘油(DAG)是具有不同生物学作用的生物活性脂质。 DAG在接受激活磷脂酶C以切割磷脂的适当刺激时在膜中瞬时累积。 所得水解产物DAG结合蛋白质例如蛋白激酶C以启动各种下游细胞过程。 DAG激酶通过将DAG转化为磷脂酸来减弱这种应答。该方案描述了设计用于定量细胞DAG水平的测定。 该测定利用DAG(1,2-二酰基甘油)到磷脂酸(1,2-二酰基-sn-甘油-3-磷酸)的酶促转化 结合DAG激酶掺入放射性标记的磷酸基团(图1)。 该测定在(Strijbis等人,2013)中描述。
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