Free flow-isoelectric focusing of human cellular lysates as sample preparation for protein analysis.
A whole cell lysate of human cells was separated into 80 fractions according to the pI 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 protein chemical 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.
D002021:Buffers; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results
D002021:Buffers; D011506:Proteins