One-dimensional SDS gel electrophoresis of proteins.
One-dimensional SDS gel electrophoresis of proteins.
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DOI：
10.1002/0471142727.mb1002as75
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发表时间：
2006-08-01
影响因子：
--
通讯作者：
Gallagher, Sean R
中科院分区：
文献类型：
--
作者：
Gallagher, Sean R
电泳法用于分离蛋白质的复杂混合物(例如，从细胞、亚细胞组分、柱组分或免疫沉淀物中分离)，研究亚基组成，并验证蛋白质样品的同质性。它还可以用来提纯蛋白质，以供进一步应用。在聚丙烯酰胺凝胶电泳法中，蛋白质在电场的响应下通过聚丙烯酰胺凝胶基质中的孔洞迁移；孔洞大小随着丙烯酰胺浓度的增加而减小。孔大小和蛋白质电荷、大小和形状的组合决定了蛋白质的迁移率。在本单元中，描述了在变性条件下，即在十二烷基硫酸钠(十二烷基硫酸钠)存在下，不连续凝胶电泳法的标准Laemmli方法。全尺寸和迷你格式都有详细介绍。针对特定的应用程序提供了几项修改。对于多肽和小蛋白的分离，用Tris-Tricine缓冲体系或在没有尿素的情况下改进的Tris缓冲体系来取代标准缓冲液。连续SDS-PAGE是一种简化的方法，凝胶和电极溶液使用相同的缓冲液，省去了堆积凝胶。其他方案包括超薄凝胶和梯度凝胶的制备和使用，以及多种凝胶的同时制备。
Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full-size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris-tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS-PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.