Stable storage conditions of Immobiline chemicals for isoelectric focusing.
Most of the problems connected with the 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 values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, 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.
Gåveby
B M
BM
Pharmacia LKB Biotechnology AB, Bromma, Sweden.
Pettersson
P
P
Andrasko
J
J
Ineva-Flygare
L
L
Johannesson
U
U
Görg
A
A
Postel
W
W
Domscheit
A
A
Mauri
P L
PL
Pietta
P
P
D000433:1-Propanol; D002021:Buffers; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004356:Drug Storage; D006868:Hydrolysis; D007525:Isoelectric Focusing; D011108:Polymers
D002021:Buffers; D011108:Polymers; D000433:1-Propanol