Most of the problems connected with use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK pathways: (a) hydrolysis of the amido bonds, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.
STABLE STORAGE-CONDITIONS OF IMMOBILINE CHEMICALS FOR ISOELECTRIC-FOCUSING / B. GAVEBY, P. PETTERSSON, J. ANDRASKO, L. INEVAFLYGARE, U. JOHANNESSON, A. GORG, W. POSTEL, A. DOMSCHEIT, P. MAURI, P. PIETTA, E. GIANAZZA, P. RIGHETTI. - In: JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS. - ISSN 0165-022X. - 16:2-3(1988), pp. 141-164.
STABLE STORAGE-CONDITIONS OF IMMOBILINE CHEMICALS FOR ISOELECTRIC-FOCUSING
E. GIANAZZA;
1988
Abstract
Most of the problems connected with use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK pathways: (a) hydrolysis of the amido bonds, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.
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