Okajima T, Tanabe T, Yasuda T
Analytical Research Laboratories of Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan.
Anal Biochem. 1993 Jun;211(2):293-300. doi: 10.1006/abio.1993.1272.
A sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system for separation of both peptides and proteins, which is a modification of the Laemmli system, is described. In the modified procedure, twofold higher concentrations of buffers in the separating gel and the running buffer solution allow superior resolution for peptides as small as M(r) 5000. The resolution of peptides was dependent on salt concentrations in the systems in which sodium chloride was partially substituted for Tris-HCl buffer and buffer concentrations were varied. In the stacking gel of the modified procedure, detection of peptides and SDS demonstrated a sharp stack of peptides at the trailing edge of the SDS stack. On the other hand, this SDS stack included peptides, forced them to diffuse, and produced a broad starting zone under Laemmli conditions. In addition, following expansion of the SDS stack impaired peptide resolution further in the separating gel. Accordingly, the different interaction with the SDS stack in the stacking process was found to produce different resolution of peptides in the electrophoretic procedures. The modified conditions have potential to provide a superior alternative to the Laemmli system for analysis of various proteins.
本文描述了一种采用不连续缓冲系统的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法，用于分离肽和蛋白质，该方法是对Laemmli系统的一种改进。在改进后的方法中，分离胶和运行缓冲液中缓冲剂浓度提高两倍，对于低至5000相对分子质量（M(r)）的肽具有更好的分辨率。肽的分辨率取决于系统中的盐浓度，其中氯化钠部分替代Tris-HCl缓冲液且缓冲液浓度有所变化。在改进方法的堆积胶中，肽和十二烷基硫酸钠（SDS）的检测显示，在SDS堆积的后缘有清晰的肽堆积。另一方面，这种SDS堆积包含肽，迫使它们扩散，并在Laemmli条件下产生较宽的起始区。此外，SDS堆积扩展后会进一步损害分离胶中肽的分辨率。因此，发现在堆积过程中与SDS堆积的不同相互作用会在电泳过程中产生不同的肽分辨率。改进后的条件有可能为分析各种蛋白质提供一种优于Laemmli系统的选择。
