Erich Gombocz

Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry.

The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.

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

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 (pIs). 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.