十二烷基硫酸钠
化学
分子质量
凝胶电泳
考马斯亮蓝
聚丙烯酰胺凝胶电泳
色谱法
二硫苏糖醇
生物化学
电泳
糖基化
钠
染色
有机化学
生物
酶
遗传学
作者
Clara L. Kielkopf,William R. Bauer,Ina L. Urbatsch
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2021-12-01
卷期号:2021 (12): pdb.prot102228-pdb.prot102228
被引量:20
标识
DOI:10.1101/pdb.prot102228
摘要
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 (β-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.
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