CS211215B1 - Method of quantitative isolation of the proteins from the polyacrylamide gels - Google Patents

Abstract

The invention relates to a quantitative method isolation of polyacrylamide proteins gels. The essence of the invention is that gel electrophoresis a cut deposited on a polyacrylamide support a buffer solution of pH 8.5 to 9.5 is added and a concentration of 0.02 to 0.2 mol / liter v amount at least equal to the goal tris / hydroxymethyl / aminomethane with addition of glycerol over which it is added layer the inorganic salt solution at the concentration 1 to 2 mol / liter in amount at least equal to the goal section volume. The importance of the invention lies in the fact that it allows crease, routine, technically unpretentious quantitative elution of goal proteins cuts, in standard electrophoretic apparatus. The invention has application t in applied research, biochemistry, immunology and hematology.

Description

The invention relates to a method for the quantitative isolation of proteins from polyacrylamide gels.

Upon detection of the protein on the polyacrylamide goal, the protein has so far been isolated either by chemical means (depolymerization with hydrogen peroxide) or by reverse polarity electrophoresis. In the chemical isolation process, a significant amount of depolymerized carrier material (gel) is present in the sample. In reverse polarity electrophoresis, it was necessary to use specially designed tubes, or modified electrophoretic devices, which used to capture the protein, e.g. dialysis membranes, or hydroxylapatite. The use of these isolation methods is associated with difficult handling and complicated preparation of individual tubes containing gel sections. The use of dialysis membranes and hydroxylapatite leads to non-specific protein uptake on these materials, resulting in non-quantitative protein isolation. In addition, the protein is captured in a large volume in most methods, which usually requires the next step of protein concentration. Application of these special tubes prevents routine processing of samples.

These disadvantages are overcome by the method according to the invention, characterized in that, prior to electrophoresis, a buffer solution having a pH of 8.5 to 9.5 and a concentration of 0.02 to 0.2 mol / liter is added to the gel cut deposited on the polyacrylamide carrier. an amount of at least equal to the gel section volume, preferably tris / hydroxymethyl / aminomethane with addition of glycerol, over which a solution of inorganic salt at a concentration of 1 to 2 moles / liter in an amount at least equal to the gel section volume is superimposed.

The ascending discontinuous conductivity gradient above the gel section prevents leakage of the protein into the electrophoretic medium, and the descending density gradient above the gel section maintains the ascending conductivity gradient.

The invention allows rapid, routine, undemanding quantitative elution of protein from goal slices in standard electrophoretic apparatuses. The eluted liquid is collected in a small volume - less than 0.2 ml.

Example

The procedure is explained in connection with the attached drawing.

A gel section containing the respective protein, e.g. myoglobin, bovine serum albumin or insulin (section B) is placed in a conventional tube which is filled up to 3/4 of its volume with a polyacrylamide gel, e.g. 10% acrylamide polymer (section A). A low conductivity solution, e.g. 0.15 ml of 0.025 M tris / hydroxymethyl / aminomethane, 0.075 M glyoin, 30% by volume glycerol (pH = 8.8) (section C). Carefully pipet above section C sodium chloride solution (section D) of high conductivity and lower density compared to the solution in section C to the top of the tube. Reverse polarity electrophoresis is performed for 1 to 3 hours (1 hour in the absence of sodium dodecyl sulfate). A current of 4 mA per tube is applied. The length of the elution depends on the type of protein isolated, the depth of the gel section and the presence of sodium dodecyl sulfate. The eluted protein is found in a small volume in the C region. with myoglobin 95%. In the case of using goal sections whose density is lower than the density of the solution in section C, the density of the section can be increased by incubating the section in a dilute solution of isopropyl alcohol.

The invention is applicable to the quantitative elution of proteins from goal slices in applied research, biochemistry, immunology and hematology.

Claims (2)

Method of quantitative isolation of proteins from polyacrylamide gels by reverse polarity electrophoresis, characterized in that before electrophoresis, a buffer solution having a pH of 8.5 to 9.5 and a concentration of 0.02 to 0.2 is added to the gel section deposited on the polyacrylamide carrier. mole / liter in an amount at least equal to the goal cut volume, preferably tris / hydroxymethyl / aminomethane with the addition of glycerol, over which a solution of inorganic salt at a concentration of 1 to 2 mol / liter in an amount at least equal to the goal cut volume is layered.