ISSN:
0173-0835
Keywords:
Buffer electrofocusing
;
Density gradient electrophoresis
;
Separation of proteins
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
A density gradient electrophoresis apparatus made of Perspex (7 cm, Ø 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 (pK1,1-1-2) and the pK2,II+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and γ-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 glycoforms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill-defined Ampholines, the cost of these well-defined amphoters is lowPresented at the “Elektrophorese Forum “96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23-25, 1996.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150180518
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