在非常酸性的固定化pH梯度中的二维图谱。

Two-dimensional maps in very acidic immobilized pH gradients.

作者信息

Sinha P K, Köttgen E, Stöffler-Meilicke M, Gianazza E, Righetti P G

机构信息

Institute of Clinical Chemistry and Biochemistry, Freije Universität Berlin, F.R.G.

出版信息

J Biochem Biophys Methods. 1990 Apr-May;20(4):345-52. doi: 10.1016/0165-022x(90)90096-u.

DOI:10.1016/0165-022x(90)90096-u

PMID:2365950

Abstract

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185-192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8-5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimension gels and for a proper silver staining of 2-D maps with practically no background deposition.

摘要

到目前为止，由于缺乏合适的可掺入聚丙烯酰胺基质中的缓冲丙烯酰胺衍生物，在非常酸性的固定化pH梯度（IPG）中进行二维（2-D）分离是不可能的。pK 3.1缓冲液（2-丙烯酰胺基乙醇酸；Righetti等人，《生物化学与生物物理方法杂志》16，1988，185 - 192）的出现使得能够配制这种酸性梯度。我们在此报告了从大鼠肠和肝细胞总裂解物中分离多肽链的IPG pH 2.8 - 5.0区间，以及从死海嗜盐菌中分离30S和50S核糖体蛋白的情况。给出了高度可重复的第一维和第二维凝胶的条件，以及用于二维图谱的适当银染条件，几乎没有背景沉积。