Abstract：
Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and β-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.
摘要：
免疫沉淀(IP)和共免疫沉淀(co-IP)是分析蛋白质表达和分子间相互作用的成熟方法。制备蛋白质的提取和洗涤缓冲液的组成对于完成实验目的很重要。缓冲液中包含各种洗涤剂以调节提取效率和洗涤效果。其中,TritonX-100(Tx-100),NonidetP-40(NP40),脱氧胆酸(DOC)和SDS通常根据实验目的和目的蛋白的特征使用。在某些情况下,一般的去污剂会破坏分子间的相互作用，使其无法分析目的蛋白与其结合配偶体的分子关系。在这项研究中,我们建议皂苷，一种天然洗涤剂,在分析脆弱的分子间相互作用时，可用于免疫共沉淀，其中肌养蛋白和肌聚糖用作代表性相互作用。本报告中最值得注意的发现之一是，在皂苷缓冲液中维持了肌营养不良蛋白和肌聚糖之间的分子间缔合，而一般的洗涤剂，如Tx-100、NP40和DOC，分离其结合。此外，补充海藻糖，它被证明是分子伴侣，有助于在co-IP测定中有效检测肌营养不良蛋白-营养不良聚糖大分子复合物。重要的是,含3%皂苷的提取缓冲液，0.5M海藻糖和0.05％Tx-100（我们将其命名为STX缓冲液）适用于另一种分子相互作用的co-IP，N-钙粘蛋白和β-连环蛋白，表明该方法可用于感兴趣的多功能蛋白质。因此，STX缓冲液是一种用于分析脆弱的分子间关联的替代提取方法，并提供了识别复杂的相互作用的机会。这可能有助于蛋白质组研究和感兴趣的蛋白质的功能分析。
