SU146086A1 - Method for investigation of protein fractions by microelectrophoresis - Google Patents

Description

The proposed method for studying protein fractions by microelectrophoresis is a modification of one of the methods of electrophoresis on a supporting medium, for example on an agar-agar gel.

The proposed method, in comparison with the known ones, allows increasing the number of simultaneously conducted studies and reducing the amount of serum per study, with the opaque background on the filter paper being replaced with a transparent agar-agar background and the electrophoresis time is reduced to 2 hours. The peculiarity of the method is that on a glass plate with agar, using a comb with strictly identical tooth sizes, rows of grooves are made into which different test liquids are introduced with a graduated pipette, an electrical current is switched on and the plate is processed by known methods.

The study of protein fractions by agar gel microelectrophoresis. May be carried out on a known apparatus, the scheme of which is shown in FIG. 1, plan view; in fig. 2 - the same apparatus, section-L in FIG. one; in fig. 3 shows a comb for performing grooves on the surface of an agar gel; in fig. 4 is a diagram of the operating position of the comb when performing grooves.

On a clean, dried, with a degreased surface glass Plate /, located in a horizontal position, pour filtered 1% agar-agar in a buffer (veronal-medinal, barantine) heated to 80 ° C and cooled to 50-60 ° the solution, whose pH is 8.6, the ionic strength is 0.08-0.1, and the optimal amount is 80 ml of the solution to obtain a layer thickness of 1.75 mm. The plate / s agar-agar is covered with a crystallization cap and cooled at room temperature 1 hour.

After cooling, the comb 2 (made of plastic) is taken, its teeth are set, having the same size, at an angle of 90 ° to the surface No. 146086

layer 3 of agar and gently press all the way into the glass plate. After 30-40 seconds, the comb is removed and the same operation is repeated in the next row of agar layer. Thus, 16 grooves 4 in one row are obtained, respectively, the number of teeth of the comb, and a total of 48 grooves in three rows, each groove has the following dimensions: 1.75 mm depth, 1 mm width and 4.5 mm length; the distance between the rows of grooves is 75 mm. The plate prepared for the work / agar is placed on the table 5 of the apparatus in a horizontal position. On the plate edges (on the agar gel) four layer wipes 6 are applied with filter paper pre-impregnated with buffer solution, by means of which the agar-agar layer is connected with buffer solution 7 filling the cuvettes 8, in which behind the partitions 9 there are anode electrode 10 and cathodic electrode // In each cuvette there is -to 900 ml of buffer solution. A graduated pipette into each groove 4 is filled with various test liquids, for example, serum or blood plasma, 0.0015-0.002 ml each (the optimal amount of protein is 0.1 milligram).

After filling all the grooves 4, the apparatus is closed with a lid 12 provided with a rubber gasket on the edges for tightness of the chamber formed in the apparatus, a leveled electrical current is connected and electrophoresis is carried out for 2 hours at room temperature (18-20). At the end of the process the plate / separated from the buffer solution (after turning off the current, the napkins 6 are removed), remove it from the apparatus and process it in the usual way. Electrophoregram staining is performed by known dyes used for staining on filter paper. The decoding of electrophoregrams is carried out on microphotometers of the type MF-2 or MF-4. Thus, it is possible to simultaneously conduct 48 experiments (studies) on conventional devices designed for electrophoresis on filter paper.

At the conclusion of the Order of Lenin of the Institute of Ge.matology and Blood Transfusion, the proposed method can be recommended for introduction into the practice of biochemical research.

Subject invention

A method for studying protein fractions using agar gel microelectrophoresis, characterized in that, in order to increase the number of simultaneous experiments, on a glass plate with an agar, a comb with strictly identical tooth sizes makes rows of grooves into which different test liquids are inserted with a graduated pipette and at the end of the process, the plate is processed by known techniques.