Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers

Electrophoresis
A TulpA A Hart

Abstract

A density gradient electrophoresis apparatus made of Perspex (7 cm, O 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glyc...Continue Reading

References

Jun 26, 1992·Journal of Chromatography·M Bier, T Long
Jun 15, 1973·Annals of the New York Academy of Sciences·H Rible
Oct 1, 1984·International Journal of Cardiology·L DalientoG Thiene

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