Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins
作者:
Angelika Görg,
Christian Obermaier,
Günther Boguth,
Adam Csordas,
Jean‐Jacques Diaz,
Jean‐Jacques Madjar,
期刊:
ELECTROPHORESIS
(WILEY Available online 1997)
卷期:
Volume 18,
issue 3‐4
页码: 328-337
ISSN:0173-0835
年代: 1997
DOI:10.1002/elps.1150180306
出版商: Wiley Subscription Services, Inc., A Wiley Company
关键词: Alkaline proteins;Histones;Immobilized pH gradient;Nuclear proteins;Ribosomal proteins;Two‐dimensional polyacrylamide gel electrophoresis
数据来源: WILEY
摘要:
AbstractBasic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two‐dimensional (2‐D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4–10 and 6–10 using a previously published protocol (Görget al., Electrophoresis1988, 9, 531–546). In the present study we have extended the pH gradient to pH 12 with IPGs 8–12, 9–12 and 10–12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2‐D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady‐state conditions. Due to the selectivity of isoelectric focusing in IPG 9–12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone ext
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