Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Treating polymer containing material or treating a solid...
Reexamination Certificate
2001-09-19
2002-10-29
Boykin, Terressa M. (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Treating polymer containing material or treating a solid...
Reexamination Certificate
active
06472503
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for drying electrophoresis gels and in particular is concerned with compositions and methods for drying polyacrylamide gels.
2. Background Art
Gel electrophoresis is a technique much used to separate biological macromolecules such as polypeptides, proteins, DNA and RNA. Polyacrylamide is one commonly used gel forming polymer.
In polyacrylamide gel electrophoresis, a polyacrylamide gel containing an appropriate buffer is cast into a thin slab between glass or plastic plates or enclosed in a glass tube. The slab is placed between electrode compartments and a small amount of a solution of the sample is pipetted into precast notches on the top of the gel. Usually a water soluble cationic or anionic tracking dye is added. The dye migrates faster than the macroions so that the progress of the experiment can be tracked. The current is turned on and run until the tracking dye band is near the bottom of the slab. The gel is then removed and generally stained with a dye that binds to the proteins or nucleic acids.
The gel cannot be stored for a prolonged period as it becomes brittle or swells excessively if left as is. Therefore, in many cases, profiles of electrophoresis gels are photographed or copied on paper or the like and stored as images.
However, in photographing or copying the gel it is difficult to accurately record thin bands or very subtle differences in density of stains. In addition, biological macromolecules which have been separated by electrophoresis cannot be recovered. In order to mitigate this problem various proposals for drying the gel for storage have been proposed.
U.S. Pat. No. 5,635,046 (Daiichi Pure Chemicals Co., Ltd.), the disclosure of which is incorporated herein by reference, describes prior art methods for drying gels.
One such method is dislosed in U.S. Pat. No. 4,883,597, the disclosure of which is also incorporated herein by reference. The patent describes a drying method in which water vapour is extracted from a gel supported by a polyethylene membrane by apply a vacuum through the membrane.
Other more complex methods involve drying devices utilizing both heat and vacuum sources in attempt to uniformly remove moisture from the gel. Such a procedure commonly involves positioning the gel matrix on a filter paper and placing the resulting combination of gel matrix on filter paper within a drying apparatus in which the combination is subjected to a vacuum source and heated to remove liquid from the gel. An example of such an arrangement is shown in U.S. Pat. No. 4,020,563. Similarly, U.S. Pat. Nos. 4,612,710 and 4,788,778 disclose a method and apparatus in which heat is provided to a horizontal gel slab from a heating plate below while drawing a vacuum beneath.
However, as pointed out in U.S. Pat. No. 5,635,046, a method in which gels are dried with heat under reduced pressure requires a dedicated drying apparatus and a vacuum pump, which are both relatively expensive. In addition, when gels contain acrylamides at high concentrations of not less than 15% by weight, frequently the gels are damaged due to cracking. Moreover, methods using an organic solvent to dewater gels tend to result in a loss in transparency or deformation of the gel, raising a problem in recording images accurately.
An alternative method for drying gels is to sandwich the gel between cellophane films (for example, extruded cellulose xanthate) or the like. An advantage of this method is that it does not require special equipment, apart from the optional use of a simple drying frame. Moreover, sandwiching the gel between two cellophane films has the further advantage of suppressing deformation of the gel during the drying process.
However, even when the cellophane sandwich method is used, cracking of the gel can occur, especially when using gels containing polyacrylamides at high concentrations of not less than 15% by weight.
Furthermore, the gels can lose transparency, particularly at low gel concentrations.
To avoid this problem, a number of methods have been proposed including incorporation of glycerol in the gel and application of gelatin or a paste onto the surface of a gel. However, these methods remain ineffective for preventing the occurrence of cracks in the case of gels having high concentrations of acrylamide.
SUMMARY OF THE INVENTION
The present inventor has found that the use of polyhydroxy alcohols in the drying process gives transparent gels that do not experience significant cracking during the dry process, even where the gels have high or low concentrations of polyacrylamide. This is a surprising outcome give that the use of glycerol, a polyol, results in significant cracking of the gel during the drying process.
Without wishing to limit the present invention in any way, it is believed that the gel cracking resulting from the use of glycerol in the drying process is in some way related to the vicinal nature of three hydroxy groups of glycerol (1,2,3 propane triol). The inventor has found that cracking of the gel during the drying process can be avoided provided that the polyhydroxy alcohol used has no vicinal hydroxy groups, or if it does have vicinal hydroxy groups, it has no more that 2 vicinal hydroxy groups.
Accordingly, the present invention provides a method for drying a polyacrylamide gel, the method comprising contacting the gel with au aqueous solution of a polyhydroxy alcohol other than a polyhydroxy alcohol having at least 3 vicinal hydroxy groups and drying the gel.
The polyacrylamide gel to be dried according to the method of the present invention is not particularly limited as long as it can be used for electrophoresis. For example, it may be a polyacrylamide gel having an arbitrary concentration or density gradient ranging from about 2 to 50% by weight. The polyacrylamide gel may be that formed from a substituted or unsubstituted acrylamide and optionally one or more other monomers.
The polyhydroxy alcohol used in the method of the invention may be any alcohol having 2 or more hydroxy groups provided that the alcohol is not one having 3 or more vicinal hydroxy groups. Preferably the polyhydroxy alcohol has a ratio of carbon to hydroxy groups such that the polyhydroxy compound remains water soluble. More preferably, the polyhydroxy alcohol has no more than 6 hydroxy groups. For example, the polyhydroxy alcohol may be a diol, triol or tetraol. Particular examples of suitable polyhydroxy alcohols are alkyl diols and alkyl tetraols. Particular example of polyhydroxy alcohols include 1,2-ethane diol, 1,2-propane diol, 1,3propane diol 1,4-butane diol, 1,6 hexane diol, 1,2,6-trihydroxy hexane, trimethylol propane and pentaerythritol. The polyhydroxy alcohol may be a polyglycerol, which is formed by the self-condensation of glycerol. The ether linkage between the glycerol units in the polyglycerol may be 1, 2 or 1, 3.
Surprisingly, it has been found that polyhydroxy alcohols in accordance with the present invention can be used in the method of the invention as an aqueous solution, without the need for an additional solvent. Not having to use additional solvents has obvious advantages in terms of environmental and safety issues. Although not required, a solvent other than water may be included in the aqueous solution. The other solvent may be any solvent used in polyacrylamide gel electrophoresis. The solvent may be such that it alters the rate of drying of the gel. The other solvent may be a water-soluble and highly volatile organic solvent. Examples of such organic solvents include C1-C4 alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and butanol, as well as solvents such as acetone, tetrahydrofuran, acetonitrile, dimethyl formamide and dimethylsulfoxide. In the present invention, methanol, ethanol, 1-propanol, and 2-propanol are preferred. The organic solvents may be used singly or in combination of two or more. It is preferred that the organic solvents be present in amounts of 0.1-80% by weight, and more preferably 1-50% by weight, in the aforementione
Baker & McKenzie
Boykin Terressa M.
Gradipore Limited
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