Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1994-03-31
1996-11-26
Tung, T.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204469, 204470, G01N 2726
Patent
active
055781802
DESCRIPTION:
BRIEF SUMMARY
size. An ideal gel system should have a reproducible pore size and no fixed charge (or at least a constant amount) and should be resistant to change in chemical characteristics or the pore size due to hydrolysis.
The size of the macromolecule varies between different macromolecules; the smaller and more compact the macromolecule the easier it will be for the macromolecule to move through the pores of a given gel. Given a constant charge density, the rate of migration of a macromolecule is inversely proportional to the logarithm of its size.
For accurate and reproducible electrophoresis, a given type of macromolecule should preferably take on a single form in the gel. One difficulty with maintaining uniformity of the shape of proteins during gel electrophoreses is that disulfide bonds can be formed by oxidation of pairs of cysteine amino acids. Different oxidized forms of the protein then have different shapes and, therefore, migrate through the gel run with slightly different mobilities (usually faster than a completely reduced protein, since the maximum stokes radius and minimum mobility should occur with a completely unfolded form). A heterogeneous mixture of forms leads to apparent band broadening. In order to prevent the formation of disulfide bonds, a reducing agent such as dithiothreitol (DTT) is usually added to the samples to be run.
The charge density of the migrating molecule is the third factor affecting its rate of migration through the gel--the higher the charge density, the more force will be imposed by the electric field upon the macromolecule and the faster the migration rate subject to the limits of size and shape. In SDS-PAGE electrophoresis the charge density of the macromolecules is controlled by adding sodium dodecyl sulfate ("SDS") to the system. SDS molecules associate with the macromolecules and impart a uniform charge density to them substantially negating the effects of any innate molecular charge.
SDS PAGE gels are usually poured and run at basic pH. The most common PAGE buffer system employed for the separation of proteins is that developed by Ornstein (1) and modified for use with SDS by Laemmli (2). Laemmli, U.K. (1970) Nature 227, 680-686. The Laemmli buffer system consists of 0.375M Tris (tris (hydroxy methyl) amino-methane), titrated to pH 8.8. with HCl, in the separating gel. The stacking gel consists of 0.125M Tris, titrated to pH 6.8. The anode and cathode running buffers contain 0.024M Tris, 0.192M glycine, 0.1% SDS. An alternative buffer system is disclosed by Schaegger and von Jagow. Schaegger, H. and von Jagow, G., Anal. Biochem. 1987, 166, 368-379. The stacking gel contains 0.75M Tris, titrated to pH 8.45 with HCl. The separating gel contains 0.9M Tris, titrated to pH 8.45 with HCl. The cathode buffer contains 0.1M Tris, 0.1M Tricine, 0.1% SDS. The anode buffer contains 0.2M Tris, titrated to pH 8.9 with HCl. For both of these systems Tris is the "common ion" which is present in the gel and in the anode and cathode buffers.
In the Laemmli system, the pH of the trailing phase in the stacking gel is about 8.9. In the separating gel, the trailing phase pH is about 9.7. At this pH, primary amino groups of proteins react readily with unpolymerized acrylamide, thiol groups are more subject to oxidation to disulfides, or reaction with unpolymerized polyacrylamide, than at neutral pH and acrylamide itself is subject to hydrolysis.
The need for uniformity and predictability is magnified in precast electrophoresis gels which are manufactured by an outside vendor and then shipped to the laboratory where the electrophoresis will be performed. Precast gels must control the properties discussed above and they must be able to maintain this control throughout shipping and storage. The shelf life of many precast gels is limited by the potential for hydrolysis of acrylamide during storage at the high pH of the gel buffer.
It is a disadvantage of a high pH gel that the polyacrylamide gel is subject to degradation by hydrolysis and has a limited shelf-life.
It is a further disadvantage
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Engelhorn Sheldon
Updyke Timothy V.
Noguerola Alex
Novel Experimental Technology
Tung T.
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