| Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from Escherichia coli
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Author:
Date:
2020-12-05
[Abstract] Skd3 (encoded by human CLPB) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial protein disaggregase that solubilizes proteins in the mitochondrial intermembrane space. This protocol overcomes the challenges associated with purifying Skd3 and allows for in depth in vitro study of Skd3 activity. Tobacco etch virus (TEV) protease is required in the purification of Skd3. Thus, we also describe how to purify high quality TEV protease for use in the purification of Skd3, other purification protocols, and in vitro assays requiring TEV ...
[摘要] [摘要] Skd3（由人类CLPB编码）是一种线粒体AAA +蛋白，由N末端锚蛋白重复域和C末端HCLR分支核苷酸结合域组成。由于在纯化蛋白质达到高质量和接近均质性方面的挑战，Skd3的功能长期未知。最近，我们描述Skd3作为人类线粒体蛋白disaggregase ，在线粒体膜间间隙增溶蛋白。该协议克服了与纯化Skd3相关的挑战，并允许对Skd3活性进行深入的体外研究。Skd3的纯化需要烟草蚀刻病毒（TEV）蛋白酶。因此，我们还描述了如何净化高质量TEV蛋白酶可用于纯化Skd3，其他纯化方案以及需要TEV蛋白酶的体外测定。
[背景] Skd3是一种线粒体AAA +蛋白，与多系统线粒体疾病VII型3-甲基谷氨酸酸尿症（MGCA7）有关（Capo-Chichi等人，2015； Kanabus等人，2015； Saunders等人，， 2015; Wortmann等人，2015 ; Kiykim等人，2016 ）。由于在体外研究Skd3的能力有限，因此对生物学功能和这些突变对Skd3活性的影响的研究仍难以捉摸（Cupo和Shorter，2020； ...
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| Preparing Viable Hippocampal Slices from Adult Mice for the Study of Sharp Wave-ripples
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Author:
Date:
2020-10-05
[Abstract] We describe a protocol for preparing acute brain slices which can produce robust hippocampal sharp wave-ripples (SWRs) in vitro. The protocol is optimized for its simplicity and reliability for the preparation of solutions, slicing, and recovery incubation. Most slices in almost every mouse prepared though the protocol expressed vigorous spontaneous SWRs for ~24 h, compared to the 20-30% viability from "standard" low sodium slicing protocols. SWRs are spontaneous neuronal activity in the hippocampus and are essential for consolidation of episodic memory. Brain slices reliably expressing SWRs are useful for studying memory impairment and brain degeneration diseases in ex vivo experiments. Spontaneous expression of SWRs is sensitive to conditions of slicing and ...
[摘要] [摘要]我们描述了一种制备急性脑切片的方案，该方案可在体外产生强大的海马锐波波纹（SWR）。该协议经过优化，其简单性和可靠性可用于制备溶液，切片和恢复孵育。通过该协议，几乎每只小鼠中的大多数切片都表现出强烈的自发性SWR，持续约24小时，而“标准”低钠切片协议的生存力为20-30％。SWR是海马的自发性神经元活动，对于巩固发作性记忆至关重要。可靠表达SWR的脑片可用于研究离体的记忆障碍和脑退化疾病实验。SWR的自发表达对记录期间的切片和灌注/充氧条件敏感。SWR的振幅和丰度通常用作可行切片的生物标记。关键改进包括快速循环，切片后较长的恢复时间（3-6小时）以及使组织在32岁时恢复°C在充分灌注的培养箱中。我们定制设备中的切片可以表达自发SWR数小时，这表明在本地网络中具有平衡的激发和抑制作用的时间很长。年龄较大的小鼠（约产后180天）的切片显示出与年龄较小的小鼠（产后21-30）相似的生存力。
[背景]急性脑切片已成为电生理学和其他神经科学研究的重要准备。尽管有大量文献描述了脑切片的用法，但通常不那么详细地讨论可靠地生产可行切片的方案。来自年长动物的切片通常不太可行。通常使用健康的细胞形态和神经元的电生理特性来描述脑切片的活力。但是，组织中看起来健康的神经元并不总是转化为正常的网络行为。例如，用标准蔗糖替代方案制备的薄片（例如，Aghajanian和Rasmussen ...
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| Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
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Author:
Date:
2020-06-20
[Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be ...
[摘要] [摘要] 丝氨酸Palmitoyltranferase （SPT）是吡哆醛5 ' 磷酸（PLP）依赖酶催化第一和限速步骤中从头合成鞘脂。SPT活动是Homeostatically调控响应水平的提高鞘脂。这SPT的稳态调节是通过小ER膜蛋白介导称为ORMDLs。在这里，我们描述了一种方法用放射性标记的L-丝氨酸以测定SPT活性的SPT活性。测定的ORMDL依赖性抑制脂质从由规定的程序被开发Hornemann 实验室。 SPT的活性也可以使用氘化的L-丝氨酸进行测定，但需要进行质谱分析，这会耗费金钱，时间和仪器。可以在细胞和无细胞系统中研究ORMDL依赖性脂质对SPT活性的抑制作用。在这里，我们提供了详细的协议来测量存在短链（C8-神经酰胺）或长链神经酰胺（C24-神经酰胺）时SPT活性。该协议的最大优势之一我们通过在HeLa细胞膜中提供外源鞘氨醇和24：1酰基辅酶A通过内源性神经酰胺合酶生成长链神经酰胺来实现这一目标。需要精密的仪器。
[背景 ] 丝氨酸palmit oyltranferase （SPT）是一种多亚基酶是在真核生物和原核生物一些广泛表达（花田等人，1997; Ikushiro 。等人，2001; Hornemann 等人，2007）.The ...
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