蛋白组学技术课件解析.pptVIP

生命科学学院发育生物学教研室 2.1 蛋白样品的提取 2.2 双向凝胶电泳技术的原理 2.3 等电聚焦的原理 2.4 SDS的原理 2.5 样品分离的策略 第一向:根据蛋白质的等电点分离 第二向:SDS 聚丙烯酰胺电泳根据蛋白质的分子量分离 2.3 等电聚焦电泳 2.3.1 等电聚焦电泳 2.3.5 Ettan IPGphor 3 第一向等电聚焦系统 Ettan DALTsix 第二向电泳系统 Ettan DALTtwelve 第二向电泳系统 参考书： 问题1: 对于一个新的组织样品，该如何进行IPG胶条选择？ 问题2: 对于低丰度的蛋白，该如何进行样品分离？ 2.5 样品分离的策略 * 介绍整个平台的组成 The pH gradient is immobilised in the PAGE, included when the gels are casted. Casted on film support, the gels are 0.5 mm thick. The pH gradient is immobilised in the PAGE, included when the gels are casted. Casted on film support, the gels are 0.5 mm thick. The 3-10 NL gradient has a plateau between pH 5 and 7 to spread out the proteins in this region compared to 3-10 L. The 3-10 NL gradient was designed to resolve serum proteins, where a lot of albumin is present and masks other spots. Cell lysates and tissue extracts are better resolved in linear gradients 3-10 (L) or 4 -9. See next slide This example of tissue extract from mouse liver shows the different spreading of spots in linear and nonlinear wide gradients. The left separation displays an even distribution over the pH range, on the right picture there is a gap of spots in the middle - the plateau between 5 and 7. SDS polyacrylamide gel: In most cases a stacking gel is not necessary for vertical separations. A smooth and straight upper surface is accomplished by overlaying the monomer solution with water-saturated butanol, or 70 % isopropanol / water just after casting and leaving the gel to polymerize under this solution layer.. Embedding with agarose overlay: When the strip has been equilibrated in SDS buffer, agarose embedding is only necessary for IsoDalt gels (we do not sell these systems any longer), because the separation is performed from the left to the right side instead of from the top to the bottom. SDS polyacrylamide gel: In most cases a stacking gel is not necessary for vertical separations. A smooth and straight upper surface is accomplished by overlaying the monomer solution wit