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The use of sigmoid p H gradients in free‐flow isoelectric focusing of human endothelial cell proteins
Author(s) -
Ma Xiao,
Wildgruber Robert,
Bauer Johann,
Weber Gerhard,
Infanger Manfred,
Grosse Jirka,
Grimm Daniela
Publication year - 2012
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.201100598
Subject(s) - isoelectric focusing , proteome , immobilized ph gradient , triosephosphate isomerase , free flow electrophoresis , chemistry , myosin , chromatography , vimentin , two dimensional gel electrophoresis , biochemistry , proteomics , biology , gel electrophoresis of proteins , polyacrylamide gel electrophoresis , enzyme , gene , immunohistochemistry , immunology
Prefractionation of proteins enhances the resolution of proteome analysis of whole cells. Free‐flow electrophoresis ( FFE ) provides a useful step in various prefractionation protocols, since matrix‐free isoelectric focusing ( FF ‐ IEF ) performed in this machine enables the enrichment of large, easily absorbable, sensitive proteins. The impact of the FFE on the success of a proteome analysis depends on the quality of the FF ‐ IEF separation procedure. Therefore, attempts are continuously being made to improve FF ‐ IEF . Here, we applied sigmoid p H gradients to the prefractionation of endothelial cell proteins. Small steps of p H incline between neighboring FFE fractions were established in p H ranges, in which the proteins of interest have their p I s. With the help of this advanced technology, we separated vimentin and cytoplasmic actin as well as triosephosphate isomerase and glyceraldehyde phosphate dehydrogenase preparatively, and found a p I of 5.9 ± 0.2 for nonmuscle myosin.
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