Free flow‐isoelectric focusing of human cellular lysates as sample preparation for protein analysis

Abstract

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.

The dynamic range of protein expression: A challenge for proteomic research

“…While this procedure has not gained widespread acceptance it should be applicable to a wide range of biological fluids and tissues. Burggraf et al [52] have also used a liquid IEF system (Octopus TM , Dr. Weber GmbH, Kirchheim Germany) to prefractionate complex protein mixtures. Using the Octopus, they were able to fractionate whole cell lysates into 80 separate pI fractions.…”

Peer Reviewed: Prefractionation Techniques in Proteome Analysis

“…A liquid curtain flowing downstream in a narrow gap between two flat glass plates coupled to an orthogonal electric field separates macromolecules as they are passively transported into 80 collection tubes. Burggraf et al used this approach to prefractionate complex sample mixtures (18).…”

A Possible Role of Intracellular Isoelectric Focusing in the Evolution of Eukaryotic Cells and Multicellular Organisms

“…An isoelectric point (pI) is such pH in which an ampholyte, i.e., a molecule with both positively and negatively charged groups (zwitterion), such as a protein, has in sum a zero charge and therefore does not move in an electric field. In a mixture of ampholytes subjected to a strong enough electric field, a pH gradient spontaneously forms in which particular species of ampholytes (including proteins) occupy distinct zones in places with pH corresponding to their isoelectric point (Svensson 1961;Burggraf et al 1995;Rilbe 1973). The presence of pH gradient has been proven to exist in a eukaryotic cell (Slavik 1983;Slavik and Kotyk 1984;Tsien and Poenie 1986;Paradiso et al 1987).…”