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Narrow‐band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed‐phase separations
Author(s) -
Zhu Yi,
Lubman David M.
Publication year - 2004
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.200305779
Subject(s) - chromatography , isoelectric point , isoelectric focusing , fractionation , chemistry , electrospray ionization , high performance liquid chromatography , mass spectrometry , electrophoresis , membrane , analytical chemistry (journal) , biochemistry , enzyme
Preparative isoelectric focusing (PIEF) is used to achieve narrow‐band fractionation of proteins from whole cell lysates of Escherichia coli ( E. coli ). Isoelectric membranes create well‐defined pH ranges that fractionate proteins by isoelectric point (p I ) upon application of an electric potential. A commercial IsoPrime device (Amersham‐Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed‐phase high‐performance liquid chromatography (NPS‐RP‐HPLC) and HPLC eluent is analyzed on‐line by electrospray ionization‐time of flight‐mass spectrometry (ESI‐TOF‐MS) for intact protein molecular weight (MW) analysis. The result is a p I versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.