Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1997-05-30
2000-07-18
Beisner, William H.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204456, 204606, G01N 2726, G01N 27447
Patent
active
060902523
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to polyacrylamide gel electrophoresis as a method for separations of proteins, peptides and nucleic acids, and primarily to SDS polyacrylamide gel electrophoresis.
BACKGROUND OF THE INVENTION
Electrophoresis is a widely used procedure for separating charged molecules, such as proteins, peptides, amino acids, nucleic acids and other macromolecules based upon the mobilities of the molecules in an electric field.
Electrophoretic separations are nearly always carried out in gels as supporting medium, the latter serving as molecular sieves that enhance separation. Polyacrylamide gel is frequently used due to its high chemical and mechanical stability. Polyacrylamide gel electrophoresis is often referred to as PAGE.
Proteins and peptides are usually denatured and treated with sodium dodecyl sulphate (SDS), an anionic detergent, prior to the electrophoresis, so-called SDS electrophoresis. Most peptides bind SDS in a constant weight ratio which give them essentially identical charge densities, and their migration velocities in a polyacrylamide gel of suitable porosity are as a consequence related to their molecular weights. Thus, prior to application to the polyacrylamide gel in SDS-electrophoresis, samples are denatured by heating in the presence of excess SDS and a thiol reagent, usually 2-mercaptoethanol or dithiothreitol.
In the earliest publications describing SDS-electrophoresis, continuous phosphate buffers were used (Shapiro, A. L., Vinuela, E., and Maizel, J. V., (1967) Biochem. Biophys. Res. Commun. 28, 815; Weber, K., and Osborne, M., (1969) J. Biol. Chem. 244, 4406). In a continuous buffer system, the same buffer of a chosen pH and ionic strength is used in the gel and in the electrode chambers.
Today, however, the technique is almost solely used with the discontinuous buffer system described in Laemmli, U. K. (1970) Nature (London) 277, 680. In discontinuous systems, the pH of the separation gel normally differs from that of the buffer. The sharpness of the sample zones may be improved by providing a "stacking gel" with higher porosity (lower polymer concentration) and significantly different pH, on top of the separation gel. In the original version used by Laemmli, the high porosity stacking gel contained 0.125 M Tris-Cl buffer, pH 6.8, while the low porosity separation gel contained 0.375 M Tris-Cl buffer, pH 8.8. The electrode reservoirs contained 0.025 M Tris, 0.192 M glycine (pH.apprxeq.8.3) and 1 gram SDS/liter. When voltage is applied to this system, glycine starts to enter the stacking gel and a sharp boundary (front) will form between a leading chloride-containing zone and a trailing glycine-containing zone. Due to the low ionisation of glycine in the latter zone (resulting pH 8.9), the passage of the front will be accompanied by a drastic increase of voltage, and peptides applied to the top of the stacking gel will concentrate in a narrow very sharp zone behind the front. When the trailing zone enters the separation gel, the pH and the mobility of glycine will increase and simultaneously protein mobilities will decrease due to the lower porosity of the separation gel. With a correctly chosen porosity of the separation gel, peptides of interest will acquire a lower mobility than glycine, "destack" and move in the separation gel with relative velocities mainly determined by their size. In today's practice it is not uncommon to use simplified versions of Laemmli's buffer system. The separation gel buffer may be used as buffer also in the stacking gel, which for a majority of the applications suffice to give sharply stacked peptide zones behind the front. In many cases it is also possible to completely omit the stacking gel and rely solely on the zone sharpening which results within the sample and in connection with sample entrance into the gel. Discussions on the use of Laemmli's discontinuous buffer system can be found in textbooks on electrophoretic techniques (Andrews, A. T., Electrophoresis, Claredon Press, Oxford 1987; Dunn, M. J., G
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Hiroko Tamura and Nobuo Ui, "New Buffer System for Disc Electrophoresis Suitable for Slightly Basic Proteins" The Journal of Biochemistry, vol. 71, No. 3 (Mar. 1972) 543-545.
M. Wyckoff et al, "Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate-Containing Buffers Using Multiphasic Buffer Systems: Properties of the Stack, Valid R.sub.f Measurement, and Optimized Procedure" Analytical Biochemistry, vol. 78, No. 2 (Apr. 1977) 459-482.
Beisner William H.
Pharmacia Biotech AB
Starsiak Jr. John S.
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