JPS58105053A - Method of two-dimentional electrophoresis - Google Patents

Abstract

PURPOSE:To improve a separating power sharply by using a plane-shaped gel having the concentration gradient of polyacrylamide and containing a buffer liquid having alkaline hydrogen ion concentration from an isoelectric point of a component to be inspected. CONSTITUTION:One surface of a glass plate 1 is processed by methacryl oxypropyltrimethoxysilane, and thereby a plane gel 2 having the concentration gradient of 4-20% and containing a buffer liquid (PH-8.6, ion strength-0.06, A liquid) composed of 5,5-diethylbarbituric acid sodium and 5,5-diethylbarbituric acid is prepared. A serum is given into a cut 3 made in the low-concentration part of the gel. Filter paper sheets 4 and 5 for liquid junction, which are impregnated with the A liquid, are connected to a negative electrode tank 6 and a positive electrode tank 7 with the A liquid put therein, and serum protein is separated by electrophoresis. The liuid junction being removed, filter paper sheets 8 and 9 for liquid junction, which are impregnated with the A liquid, are connected to a negative electrode tank 10 and a positive electrode tank 11 with the A liquid put therein, and the serum protein is separated according to the difference in molecular weight.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for analyzing body fluids such as blood for the purpose of clinical testing, and in particular to a two-dimensional electrophoretic analysis method suitable for simultaneous multi-component analysis of various proteins contained in body fluids. It comes out.

Conventionally, in the field of clinical chemistry transposition of blood carcass body fluids, a one-dimensional tortoise phoresis method using a membranous material such as cellulose acetate as a support for total phoresis has been routinely performed. However, only about five types of serum proteins can be analyzed at the same time, which cannot meet the demand for an increase in the number of test items accompanying recent advances in medicinal chemistry. In this conventional electrophoretic separation method, component proteins are separated based on averaged differences in their properties such as charge amount, molecular weight, and molecular shape.
Separation power is reduced.

An object of the present invention is to improve the drawbacks of the conventional methods described above, and to provide a simple two-dimensional electrophoretic separation method for proteins that allows simultaneous analysis of multiple components. This electrophoretic separation method d is a method in which proteins that have been separated according to the principle of conventional methods are dramatically separated based on the difference in molecular weight, and by this method, the separation ability has been greatly improved.

In the present invention, two-dimensional electrophoresis is easily carried out using the same flat polymer gel without distinguishing between the supports used for first-dimensional and second-order round electrophoresis.

That is, the present invention provides a mechanical strength trading substrate such as glass or polyester with a 7-rank cassoplating agent (r).
ts plate x3, R is a vinyl group, X is an ethoxy group, and a gel having a concentration gradient of acrylamide in one direction is bonded onto this substrate. , fix the gel, add the sample to be analyzed to the low concentration part of the gel, apply electricity in the direction of the constant concentration of the gel, perform the first-dimensional electrophoretic separation, and then apply electricity in the direction of the Kel concentration gradient of this flat plate. Separation is performed based on molecular weight differences using the gel's molecular sieve effect.

Prior to electrophoresis, a flat cell used as a support for electrophoresis is preliminarily impregnated with a buffer solution having a hydrogen ion concentration more alkaline than the isoelectric point of the protein component to be separated. It is preferable to use a loose sleeve liquid and a water bath liquid having a pH of 8.6 to 8.8 and an ionic strength μ of 0.05 to 0.10.

Currently, 111 mL of one-dimensional electrophoresis of serum proteins is added to this buffer.
fK is also commonly used in clinical examinations. Using this buffer, precise comparisons between the method of the present invention and conventional medical techniques are facilitated (3). However, in other examples, similar results can be obtained even if a buffer solution consisting of borate is used.

An embodiment of the present invention will be described below with reference to the drawings.

As shown in FIG. 1, one side of a 11 crn square glass plate 1 is treated with methacryloxypropyltrimethoxysilane, and the acrylamide concentration gradient is 4 to 20% on 10 crn square sides on the glass plate. - Buffer solution consisting of sodium diethylbarbylate and 5°5-diethylbarbylic acid (pH 8.6, ionic strength 0.0)
6. A flat plate 2tl having a thickness of 2rIrIn containing liquid A) was prepared. The arrow in the figure shows the concentration gradient from the low-concentration side of Bacrylamide Kel to the high-definition side 1 (the same applies to Figure 2). A three-square groove 3 is made in the lower thick part of the gel and at the end of the gel, and 10 μm of serum is added into this groove.

Next, A'g! A filter paper 4 for liquid junction impregnated with L is placed as shown in FIG. 1, and connected to a negative electrode tank 6 also filled with liquid A. Similarly, the liquid filter paper 5 impregnated with liquid A is connected to the positive electrode tank 7 which also contains liquid A, and (4) electricity is applied between both electrodes to electrophoretically separate serum proteins (in the first dimension). Minute II 1.). Next, this liquid junction is removed, and the liquid junction filter paper 8 impregnated with liquid A is placed at the low concentration end of the gel as shown in FIG. do. Similarly, the liquid filter paper 9 impregnated with liquid A is connected to the positive electrode tank 11 containing liquid A, and current is applied between both electrodes to separate serum proteins based on the difference in molecular weight.

As described above, two-dimensional electrophoretic separation was easily performed using the same flat gel plate. Instead of directly handling the gel containing the separated proteins, the glass substrate was handled and immersed in a mixture of the dye Komatsushi Blue R, -250 in nathanol acetate.
Next, this Kel was immersed in an acetic acid aqueous solution to remove the dye present in the part of the Kel that did not contain protein.
Separated spots of 0 types of proteins could be detected.

As described above, the glaze according to the present invention was able to separate about 8 times as many types of components as the 5 glazes, which was conventionally obtained using a cellulose acetate membrane.

In the present invention, for the first-dimensional separation, a migration method similar to that used in conventional clinical tests is used, so there is a strong correlation between the analytical results of the conventional method and the analytical results of the present invention.
r: It is a temple. Therefore, the analytical approach according to the present invention has the characteristic of reinforcing the approach of the conventional method, and improves diagnostic accuracy. That is, when the protein amount (proportional to the amount of adsorbed Comatsea Blue) of each separation spot on the two-dimensional electrophoretic separation image in the present invention is projected onto the -dimensional electrophoretic separation axis, (
(This can be done by image processing of a two-dimensional electrophoretic image using an 11-machine computer), it is possible to reconstruct the separation take by the conventional method.

[Brief explanation of the drawing]

1 and 2 are plan views of a two-dimensional electrophoresis apparatus for carrying out the method of the present invention. DESCRIPTION OF SYMBOLS 1... Substrate, 2... Support for electrophoretic separation, 3... Groove for adding test sample, 4... Liquid junction, 5... Liquid junction, 6...
・Negative electrode tank, 7...Positive electrode tank, 8...Liquid junction, 9...
・Liquid junction, 10...Negative electrode tank, 11...Positive electrode tank Agent Patent attorney Toshiyuki Usuda

Claims (1)

[Claims] 1. As a support for electrophoretic separation, a concentration gradient plate of polyacrylamide containing a buffer solution with a concentration of hydrogen ions more alkaline than the isoelectric point of the test component and with the negative side fixed to the substrate was used. A two-dimensional electrophoresis method characterized by adding the test sample to the low-density part of the non-fixed surface of the cell and further separating the sample components.