Carrier ampholytes rehabilitated: gel isoelectric focusing on pH gradients visualized in real-time by automated fluorescence scanning in the HPGE-1000 apparatus.

Abstract

All synthetic carrier ampholyte mixtures (SCAMs) contain some naturally fluorescing carrier ampholytes (CAs). The detection of these during isoelectric focusing (IEF), using gel electrophoresis apparatus with intermittent scanning of fluorescence, allows one to follow in real-time the "life cycle" of the pH gradient, i.e., its genesis, steady-state, and decay. The most prominently fluorescing CAs can be calibrated by pH measurement at or after the steady state ("calibration CAs"). By application of the calibration CAs, the fluorescence pattern of CAs can be interpreted in terms of pH gradient (p/s). Simultaneously with the visualization of the pH gradient in that way, protein samples can be detected by "fluorescence reduction" and assigned pH values in dependence on focusing time and, at or after the steady state of the protein, pI' values. The method remedies the inherent blindness of IEF with regard to the state of the pH gradient within its limited "life cycle". It allows one to load the sample at a time when the shape of the pH gradient is optimal for the purpose of its resolution from neighboring components. The visualization of the cathodic drift during IEF eliminates the danger to resolution and to loss of sample associated with "blind" IEF. Most importantly, the possibility to follow the pH in the position of the protein as a function of time provides an objective, accurate measure of the pI' not available from pH measurement at an arbitrary focusing time. The method therefore preempts the advantage of using an IEF method which is free of the pH gradient drift, i.e., immobilized pH gradient (IPG)-IEF. Moreover, it preserves the "natural pH gradient", does not present any of its sample entry problems and those due to very low conductance, and is compatible with agrose gels and their relatively diminished restrictiveness to migration.