什么是电泳?其影响因素有哪些? SDS-PAGE技术有何特点和用途?
推荐回答(2个)
带电颗粒在电场作用下,向着与其电性相反的电极移动,称为电泳。
聚丙烯酰胺凝胶电泳 作用:用于分离蛋白质和寡核苷酸。
作用原理 聚丙烯酰胺凝胶为网状结构,具有分子筛效应。它有两种形式:非变性聚丙烯酰胺凝胶及SDS-聚丙烯酰胺凝胶(SDS-PAGE);非变性聚丙烯酰胺凝胶,在电泳的过程中,蛋白质能够保持完整状态,并依据蛋白质的分子量大小、蛋白质的形状及其所附带的电荷量而逐渐呈梯度分开。
而SDS-PAGE仅根据蛋白质亚基分子量的不同就可以分开蛋白质。该技术最初由shapiro于1967年建立,他们发现在样品介质和丙烯酰胺凝胶中加入离子去污剂和强还原剂后,蛋白质亚基的电泳迁移率主要取决于亚基分子量的大小(可以忽略电荷因素)。
SDS是阴离子去污剂,作为变性剂和助溶试剂,它能断裂分子内和分子间的氢键,使分子去折叠,破坏蛋白分子的二、三级结构。而强还原剂如巯基乙醇,二硫苏糖醇能使半胱氨酸残基间的二硫键断裂。在样品和凝胶中加入还原剂和SDS后,分子被解聚成多肽链,解聚后的氨基酸侧链和SDS结合成蛋白- SDS胶束,所带的负电荷大大超过了蛋白原有的电荷量,这样就消除了不同分子间的电荷差异和结构差异。
SDS-PAGE一般采用的是不连续缓冲系统,与连续缓冲系统相比,能够有较高的分辨率。
浓缩胶的作用是有堆积作用,凝胶浓度较小,孔径较大,把较稀的样品加在浓缩胶上,经过大孔径凝胶的迁移作用而被浓缩至一个狭窄的区带。当样品液和浓缩胶选TRIS/HCL缓冲液,电极液选TRIS/甘氨酸。电泳开始后,HCL解离成氯离子,甘氨酸解离出少量的甘氨酸根离子。蛋白质带负电荷,因此一起向正极移动,其中氯离子最快,甘氨酸根离子最慢,蛋白居中。电泳开始时氯离子泳动率最大,超过蛋白,因此在后面形成低电导区,而电场强度与低电导区成反比,因而产生较高的电场强度,使蛋白和甘氨酸根离子迅速移动,形成以稳定的界面,使蛋白聚集在移动界面附近,浓缩成一中间层。
摘自百度百科,你可以自己查看百度百科,或者去CNKI里检索的看一下
电泳是利用物质带电性质而在电场下泳动的特性从而对物质进行分离的技术。
影响因素主要是物质的带电荷的多少,物质的分子量大小,物质的空间形态等。
SDS-PAGE主要用于变性蛋白质的分离和鉴定,主要特点是蛋白经SDS处理后均匀带电荷并形成线性的结构,从而在聚丙烯酰胺胶中的泳动只与蛋白的分子量大小有关。
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