CS261027B1 - Process for preparing a polyacrylamide gel especially for electrophoresis - Google Patents

Abstract

The solution concerns the field of polymers and analytical chemistry. The technical problem of preparing polyacrylamide gels for electrophoresis is solved. The essence of the solution is to carry out the polymerization of a 3.5 to 28% solution of acrylamide and N,N'-bis-methyleneacrylamide in a molar ratio of 1 to 0.01 to 0.05 in the presence of sodium hydrosulfite dihydrate and methylene blue by the action of potassium or ammonium persulfate. The solution can be used in the preparation of gels for electrophoretic separations of polypeptides, proteins and other charged particles in general.

Description

The invention relates to a process for the preparation of polyacrylamide gel, in particular for electrophoretic separation.

Electrophoretic separation of polypeptides, proteins, enzymes, and other electrically charged particles is widely used in basic and applied research in biological systems. The separation is usually carried out in an aqueous gel layer based on agar, agarose, starch, modified cellulose or polymeric acrylamide. Relatively best separation results are obtained especially on polyacrylamide gels cross-linked with, for example, Ν, Ν'-bis-methyleneacrylamide. This gel is prepared without major inconvenience even in laboratory conditions, it is sufficiently mechanically strong and can achieve a satisfactory separation effect. A serious drawback, however, is the fluctuating quality of the gel prepared according to the current state of the art by radical polymerization, most often by the action of persulfate and tertiary nitrogen containing compounds. In part, these problems can be reduced by using highly pure components of the polymerization system, but the main cause of the lack of reproducibility - inhibition of polymerization by air oxygen - remains.

The state of the art does not know how to achieve the reproducibility of the gel quality that is necessary for electrophoretic separation (except for polymerization in an inert medium which is technically very demanding).

Based on experimental work, we have found a new process for the preparation of polyacryamide gel which eliminates the above mentioned disadvantages caused by diffusion of air oxygen and allows to prepare gels for electrophoretic separation with very good reproducibility. The process for preparing a polyacrylamide gel according to the invention consists in mixing a 3.5 to 28% aqueous solution of 100 parts by weight of acrylamide and Ν, Ν'-methylene-bis-acrylamide in a molar ratio of 1 to 0.01 to 0.05 with The pH of the solution is adjusted to 8-9 by adding 1.0 to 10.5 parts by weight of a 0.5% methylene blue solution and adding a buffer containing 0.1 to 0.4% of Ν, Ν, Ν ', Ν'-tetramethylethylenediamine. the solution is treated with a 0.4 to 1.0% sodium bisulfite solution to discolor. 0.2 to 0.6 parts by weight of sodium bisulfite dihydrate powder was added to the colorless solution, and the homogeneous solution was treated with 500 parts by weight of 0.1 to 0.5% potassium or ammonium persulfate.

At room temperature, polymerization to the gel stage takes place within no more than five minutes and reaches the full density of the polymer network after 2 to 12 hours, depending on the temperature.

The method according to the invention provides a very reproducible quality of the gels, which is best demonstrated by the electrophoretic separation. The application of other redox acrylamide polymerization systems, such as persulphate-sulfite, persulphate-sulphate, etc., does not match the technical effect of the polymerization process of the invention. The redox system of the invention is highly reactive in the preparation of aqueous gels and concurrently eliminates the undesirable effect of oxygen inhibition of polymerization, which is uncontrolled and unreproducible with known consequences in the case of other initiation systems. The color indicator used allows precise control of the redox potential of the polymerization medium, the failure of which will immediately become bluish. A significant advantage of the process according to the invention is that once the polymerization has been completed, the physiological redox potential of the environment is again achieved in the gel due to the hydrosulfite instability. Undesirable substances (electrically charged) are easily removed by pre-run electrophoresis.

Equally important advantages of the present process include the achieved sharper separation of substances with very good reproducibility.

Example

Prepare the following pure solutions in advance:

solution A:

28.0 g of acrylamide and 0.735 g of Ν, Ν'-methylene-bis-acrylamide in 100 ml of deionized water, kept at 0-4 ° C.

solution B:

Dissolve 36.6 g of tris- (hydroxymethyl) aminomethane and 200 µl of Ν, Ν, Ν ', Ν'-tetramethylethylenediamine in · 40 ml of deionized water, adjust the pH of the solution to 8.9 with 1N HCl and make up the solution with deionized water. total volume 100 ml.

solution C:

200 mg of sodium bisulfite dihydrate in 25 ml of deionized water.

solution D:

100 mg potassium persulfate in 50 ml deionized water.

Method:

Mix 6 ml of solution A with 3 ml of solution B and dilute with 10 ml of deionized water. The solution is stained with 1 drop of a 0.5% methylene blue solution with stirring. Solution C is added dropwise to the colorless solution, 7 mg of sodium bisulfite dihydrate powder are added to the colorless solution and, after dissolution, 5 ml of solution D are stirred. is poured into the space of the electro foretic plate or tube. After 2 to 5 minutes, the solution is gelled and aged at 25 ° C for 12 hours.

Claims (1)

A process for the preparation of a polyacrylamide gel, in particular for electrophoresis, by redox polymerization of acrylamide and Ν, Ν'-bis-methylene acrylamide in aqueous medium in the presence of a buffer, characterized in that a 3.5-28% aqueous solution of 100 weight parts acrylamide and Ν, Ν bis-methylenacrylamide in a molar ratio of 1 to 0.01 to 0.05 is mixed with 1.0 to 10.5 parts by weight of a 0.5% methylene blue solution and the addition of a buffer containing 0.1 to 0.4% Ν, Ν, The pH of the solution is adjusted to 8-9 with působí ', N'-tetramethylethylenediamine, then treated with 0.4-1.0% sodium bisulfite solution to decolorize, 0.2 to 0.6 parts by weight of powdered solution are added to the colorless solution. sodium bisulfite dihydrate, then the homogeneous solution is treated with 500 parts by weight of potassium or ammonium persulfate at a concentration of 0.1 to 0.5% until the polymerization is complete.