| Histone Deubiquitination Assay in Nicotiana benthamiana
|
|
Author:
Date:
2018-03-05
[Abstract] Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo assay for histone deubiquitination activity. After infiltrating UBP12 into Nicotiana benthamiana leaves, H2Aub was visualized by immunocytochemistry. Nicotiana benthamiana leaves, which show high agro infiltration efficiency, were used for transient UBP12 expression for a labor- and time-saving protocol. Reduced H2Aub levels indicated histone deubiquitination activity of UBP12. The clear visualization of nuclei of N. ...
[摘要] 组蛋白修饰是一组组蛋白翻译后修饰，可以改变染色质结构并影响基因表达。组蛋白泛素化是组蛋白H2A和H2B特异性发现的组蛋白修饰。泛素特异性蛋白酶（UBP）可以逆转组蛋白泛素化。在这里，我们描述了组蛋白去泛素化活性的体内试验。在将UBP12渗入烟草叶片中后，通过免疫细胞化学观察H2Aub。表现出高的农业渗透效率的本氏烟草叶用于瞬时UBP12表达，用于节省劳力和时间的方案。 H2Aub水平降低表明UBP12的组蛋白去泛素化活性。 N的核的清晰可视化。本生叶使得该方法能够通过使用特异性抗体容易地测量体内组蛋白修饰的水平，从而提供强大的蛋白质功能线索。因此，该协议是组蛋白去泛素化活性的体外试验的有力补充。
【背景】组蛋白修饰在调节染色质结构和基因表达中发挥重要作用。 研究最深入的组蛋白修饰包括甲基化，乙酰化，磷酸化，泛素化和sumoylation。 然而，引入或去除特定组蛋白修饰的酶并不总是已知的。 强大的体外试验可以确定组蛋白修饰酶的催化潜能，但是体内试验方法对于确认体外试验的特异性反映抗体的特异性是必要的。 体内活动。 在这里，我们描述了一个灵活的协议来测试植物组织中组蛋白修饰酶的活性。本塞姆氏。 尽管我们使用该方案来测试遍在蛋白特异性蛋白酶（UBP）对泛素化H2A的活性，但它也可以容易地用于其他特异性抗体可用的组蛋白修饰。
|
|
|
| Determination of the H+-ATP Synthase and Hydrolytic Activities
|
|
Author:
Date:
2016-08-20
[Abstract] The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we ...
[摘要] 内线粒体膜的H sup + -ATP合酶利用呼吸链产生的质子梯度来合成ATP。 在去极化条件下，它可以通过水解ATP产生质子梯度而反向作用。 本文提供的方案允许容易地测定分离的线粒体和透化的哺乳动物细胞中H sup + -ATP合酶的合成和水解活性。 由于该协议需要极化和良好耦合的线粒体的分离，首先我们描述线粒体从小鼠组织分离的协议。 第二，我们描述了用于测量ATP合成活性作为终点和在分离的线粒体和透化细胞中的动力学模式的方案。 最后，我们描述了用于测定酶在分离的线粒体中的ATP水解活性的方案。
|
|
|
| Isolating Liver Mitochondria by Differential Centrifugation
|
|
Author:
Date:
2016-05-20
[Abstract] In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
[摘要] 除了旨在研究线粒体功能的方法之外，充分了解线粒体功能需要在特定生理或病理条件下从细胞或组织中纯化。 该方案说明从小鼠肝组织获得高产量的功能性线粒体的顺序程序。 用该方法获得的线粒体可用于评估不同线粒体参数，包括氧消耗，膜电位和钙保留能力。
|
|
|