Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
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Author:
Date:
2020-12-05
[Abstract] Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were ...
[摘要] [摘要]蛋白质间相互作用 在核过程中起关键作用,包括转录,复制,DNA损伤修复和重组。免疫共沉淀(Co-IP),然后进行蛋白质印迹或质谱分析是鉴定蛋白质-蛋白质相互作用的宝贵方法。在Co-IP中定位于细胞核的蛋白质中的挑战之一是从亚核级分中提取核蛋白质,而又不会失去生理上相关的蛋白质相互作用。在这里,我们描述了一种用于天然Co-IP的协议,该协议最初用于成功地识别以前称为新拓扑拓扑异构酶1(TOP1)相互作用的蛋白质。在此协议中,我们首先通过依次增加去污剂和盐浓度来提取核蛋白,然后将提取的级分稀释,合并并用于Co-IP。该协议可用于鉴定多种哺乳动物细胞中其他染色质相关蛋白的蛋白相互作用组。
背景]钴- IP被广泛地被使用,以解开的错综复杂的关系之间的蛋白复合物和各种染色质交易期间的复制,转录,和基因组的维护。但是,它是具有挑战性的,以保持不稳定的蛋白质-蛋白质相互作用的完整过程中提取,免疫沉淀和一个共同的IP实验的洗涤步骤。稳定不稳定蛋白质相互作用的一种方法是在细胞裂解之前用细胞可渗透的可逆化学交联剂(例如丙酸二硫代双琥珀酰亚胺酯)处理细胞(Smith等人,2011)。由于该方法伴随着诸如提取效率低和非特异性蛋白质捕获之类的缺点,因此优选不交联的Co-IP(天然IP)。
一核蛋白质可以被分配到不同的子-核舱或染色质区域是需要不同程度的严格性为它的提取和溶解。对于例如,TOP ...
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Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes
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Author:
Date:
2018-01-20
[Abstract] Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture of multiple types of stem/progenitor cells, we recently established a novel method to generate bipotent liver progenitor cells, named chemically induced liver progenitors (CLiPs), from adult rat MHs. Here, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat MHs and then describe how to induce CLiPs from these MHs. In addition, we describe a method to evaluate the bipotentiality of ...
[摘要] 原代成熟肝细胞(MH)或其祖细胞是重症肝病中细胞移植治疗的候选细胞来源。然而,这些细胞的稳定培养或多能干细胞的等效细胞的产生受到限制。我们使用先前在多种类型的干/祖细胞稳定培养中发现有用的小分子混合物,最近建立了一种从成年大鼠MHs产生双能肝脏祖细胞(命名为化学诱导肝祖细胞(CLiPs))的新方法。在这里,我们描述了诱导大鼠CLiPs的详细方案。我们首先描述分离原代鼠MH的方法,然后描述如何从这些MH中诱导CLiPs。另外,我们描述了一种评估产生的CLiPs分化成肝细胞和胆管上皮细胞的双能性的方法。我们还介绍了如何通过长期的文化和详细的示例数据建立稳定的CLiP。可以在2周内产生初级CLiPs,并且可以在2.5-4个月内建立经历10次传代的稳定的CLiPs,批次间变异性。 【背景】对于实现肝病再生医学的新型细胞来源有着强烈的需求。目前唯一的治疗终末期肝病的方法是肝移植,但是由于供者短缺,其应用受到限制。最近,我们小组提出了一种产生能够在体外稳定地扩增的新型LPC的方法,并且可以以广泛的效率重新繁殖慢性肝炎动物模型的损伤肝脏(Katsuda等人, / ...
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