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Free flow‐isoelectric focusing of human cellular lysates as sample preparation for protein analysis
Author(s) -
Burggraf Dorothe,
Weber Gerhard,
Lottspeich Friedrich
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601169
Subject(s) - isoelectric focusing , chromatography , free flow electrophoresis , chemistry , coomassie brilliant blue , lysis , resolution (logic) , sample preparation , electrophoresis , two dimensional gel electrophoresis , silver stain , isoelectric point , polyacrylamide gel electrophoresis , gel electrophoresis of proteins , proteomics , biochemistry , microbiology and biotechnology , biology , staining , enzyme , artificial intelligence , computer science , gene , genetics
A whole cell lysate of human cells was separated into 80 fractions according to the p I of proteins using free flow isoelectric focusing with carrier ampholytes. The resolution of the process was highly reproducible, with an overlap of fractions of less than 30%. A protein of a faint silver stained spot in two‐dimensional gel electrophoresis (2‐DE) could be enriched, yielding a Coomassie blue stained spot which could be further characterized by proteinchemical methods. The enrichment of less abundant proteins from a complex crude cell extract was found to be a suitable tool for sample preparation and enrichment before applying proteins to 2‐DE and reversed‐phase high performance liquid chromatography.