Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes
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Author:
Date:
2020-11-20
[Abstract] Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches. We have used this technique successfully to purify both GFP-tagged and mCherry-tagged proteins, and their interacting partners, expressed in Drosophila melanogaster embryos. Although we demonstrate the efficacy of the GFP-binding protein and RFP-binding protein nanobodies from Chromotek, in theory any antibody ...
[摘要] [摘要]裂解亲和纯化(Cl-AP)使用蛋白质A-链霉亲和素珠和纳米抗体的三方体系,再加上生物素化的,硫醇可裂解的接头,可从表达标记蛋白的组织裂解物中一步纯化。这项技术可以从细胞中完整分离出荧光形式的有丝分裂蛋白复合物,用于生物物理和基于显微镜的分析中,克服了还原论方法的传统局限性。我们已经成功地使用了该技术来纯化在果蝇中表达的GFP标记和mCherry标记的蛋白及其相互作用的伴侣。 胚胎。尽管我们证明了Chromotek的GFP结合蛋白和RFP结合蛋白纳米抗体的功效,但从理论上讲,任何抗体都可以偶联至磁珠并用作Cl-AP试剂。
[背景技术]许多蛋白质引起它们的细胞功能的多蛋白复合物的一部分。为了全面了解蛋白质复合物的作用,需要将体内方法(例如干扰蛋白质水平/活性或监测动态定位)与体外生化测定和功能重建结合起来。目前,这种整体方法受到严重限制。体外研究通常使用已在非自体系统(例如细菌和昆虫细胞)中单独表达和纯化的蛋白质,其中对于功能至关重要的折叠和翻译后修饰可能与原始细胞中发现的不同。相反,从细胞/组织中纯化特定蛋白质或复合物通常依赖于共免疫沉淀或纯化已引入细胞的目标蛋白质的标记版本,例如使用血凝素(HA),FLAG 3或串联亲和纯化(TAP)标记。这些体内方法存在两个主要问题:(i ...
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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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Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network
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Author:
Date:
2020-03-05
[Abstract] Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions.
[摘要] [摘要] 反式高尔基体网络(TGN)上的蛋白质分选在将新合成的蛋白质靶向其特定目的地方面起着重要作用。该提议的目的是利用大鼠肝细胞溶质,半完整的哺乳动物细胞和核苷酸,在TGN处将非高尔基驻留的货物蛋白重新包装成囊泡。该协议描述了如何进行囊泡形成测定,如何分离囊泡以及如何检测囊泡中的货物蛋白。该重构测定法可用于定量测量在特定实验条件下将特定货物蛋白包装到TGN的运输小泡中的效率。
[背景] 的反式高尔基体网络(TGN)是在分泌运送路径的必要的交通枢纽。为了确保水泡运输的保真度,真核细胞利用各种蛋白质分选设备将特定的货物蛋白质准确地包装到TGN的运输小泡中,然后运至特定的目的地(Guo 等人,2014)。为了加深我们对TGN分选过程特异性的理解,重要的是开发一种能够忠实地重构TGN囊泡形成和货物分选过程的分析方法。该测定法可用于直接和定量地测量特定因子在调节特定货物蛋白包装到运输小泡中的作用。从内质网(ER)将货物蛋白包装到COPII囊泡中的无细胞重构已得到很好的建立(Kim 等,2005; Kim 等,2007; Merte 等,2010; Yuan 等。,2018;Niu 等,2019;)。已经开发出一种体外测定法,其在TGN处重构特定货物蛋白TGN46在运输小泡中的释放(Ponnambalam 等,1996;Wakana ...
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