Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Proteins.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Proteins.
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DOI：
10.1101/pdb.prot102228
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发表时间：
2021-12-01
影响因子：
--
通讯作者：
Urbatsch, Ina L
中科院分区：
文献类型：
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作者：
Kielkopf, Clara L;Bauer, William;Urbatsch, Ina L
大多数蛋白质分析电泳是通过聚丙烯酰胺凝胶中的分离来实现的，条件是确保蛋白质解离成单个多肽亚基并最大限度地减少聚集。最常见的是，阴离子去污剂十二烷基硫酸钠 (SDS) 与还原剂（β-巯基乙醇或二硫苏糖醇）结合使用，并在上样到凝胶之前加热解离蛋白质。 SDS 结合使多肽变性并产生负电荷，掩盖其固有电荷。 SDS 结合量通常与序列无关并且与分子量成正比；饱和时，每两个氨基酸大约结合一个 SDS 分子，或每克多肽结合 1.4 g SDS。因此，SDS-多肽复合物在电场中的迁移与多肽链的相对大小成正比，并且可以通过与已知分子量的蛋白质标记物比较来估计其分子量。然而，疏水性、高电荷序列和某些翻译后修饰（例如糖基化或磷酸化）也可能影响迁移。因此，修饰蛋白质的表观分子量并不总是准确反映多肽链的质量。该方案描述了 SDS-PAGE 凝胶的制备和运行，然后使用考马斯亮蓝染色来检测蛋白质。最后，染色的 SDS-PAGE 凝胶可被扫描成图像或通过干燥保存。
Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (beta-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or 1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.