JPS58140634A - Simple two-dimensional electrophoresis method - Google Patents

Abstract

PURPOSE:To prevent the damage of gel and to simplify an operation, by sticking concentration gradient gel on one substrate having high mechanical strength and a constant concentration gel of low concentration to the low concentration side of said gel leaving a fixed gap and then, performing two-dimensional migration separation by one substrate. CONSTITUTION:A two-dimensional migration gel 3 in which N(CH2OH)3 and an aqueous solution of glycine (A solution) are incorporated to polyacrylamide concentration gradient gel, is formed on a substrate 1 having high strength such as glass. Next, one- dimensional migration gel 2 having constant concentration such as the polyacrylamide is formed leaving a gap 4. A slit 5 for adding a sample (blood serum etc.) is cut on the gel 2 and said gel is connected to negative electrode bath 7 and positive electrode bath 9 respectively by filter papers 6, 8 for liquid connection. Then, the sample is added and it is separated by the difference of an isoelectric point. Next, the filter papers 6, 8 are removed and said solution A is held in the gap 4. Further, the gel 2 and 3 are connected to each negative electrode bath 11 and a positive electrode bath 13 by filter papers 10, 11 for liquid connection and protein is separated by the difference of molecular weight. In this manner, an operation is simplified and separation capacity is improved without breaking down the gel films 2, 3.

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 is something.

A two-dimensional electrophoretic analysis method using a polyacrylamide gel as a support, which has been conventionally used as a method for simultaneously analyzing multiple components of blood proteins (for details, see "Protein/Nucleic Acid-Enzyme", Kyoritsu Shuppan, September 1978 issue 211) (see page),
First, a long, slender cylindrical gel containing an ampholyte is created in a glass tube, the gel tube is held vertically, a voltage is applied to both ends of the gel, and component proteins in a blood sample added to one end of the gel are heated to their isoelectric point Separation based on the difference in (first-dimensional separation).

Next, at the low concentration end of a flat gel having a concentration gradient of acrylamide prepared between two glass plates (inside the gel holding frame), the same cylindrical shape with which the first dimension separation has been completed is placed. The gel is pushed out from the glass tube, the gel plate is set vertically, and electricity is applied again in the direction of the concentration gradient of the flat gel to perform separation based on the difference in molecular weight at the two-dimensional port. Next, in order to quantify each separated protein, the two glass plates sandwiching the flat gel are removed, and the gel is immersed in a dye solution such as Komatsu sea blue to stain the protein, and the background is removed. Stain and measure the absorbance of the remaining stained protein spots.

However, the elongated gel used in the first dimension is soft, and it is difficult to extrude it from a glass tube and bring it into close contact with one end of the flat gel without any gaps using a mechanical method. Unfortunately, it is difficult to remove the soft flat gel after two-dimensional electrophoresis from between the glass plates and handle it during a series of operations such as staining, destaining, and absorbance measurement due to problems such as damage to the gel.

An object of the present invention is to provide a simple two-dimensional electrophoresis method with good operability that overcomes the drawbacks of the conventional methods described above.

In the present invention, a support used for electrophoretic separation of first and second dimensions is attached adjacent to a substrate with high mechanical strength,
Two-dimensional electrophoretic separation is performed using this single substrate.

That is, a substrate such as glass or polyester is treated with a silane coupling agent (R8iXs, nr! A compound consisting of an organic residue such as a vinyl group and X is an ethoxy group, for example), and on this substrate, A polymer gel with a concentrated acid gradient of acrylamide is bonded and fixed. Next, at the low concentration end of this concentration gradient gel, at a certain interval, the concentration of acrylamide is constant, and the
Low concentration gels are also bound and identified. The present invention performs first-dimensional electrophoretic separation using a gel with a constant gel concentration as a support among the gels obtained in this way, and the analyte components separated in this gel are pre-contained with electrolytes. The gel is separated by moving an electric body through the gap between the two gels, which have been left to stand for a while, to the concentration gradient gel shell.

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

One side of the glass plate 10 is treated with methacryloxypropyltrimethoxysilane, and there is an acrylamide concentration gradient of 4 to 20% on each side of the glass plate 10, and 0.05%.
M of trishydroxymethylaminomethane and “0.
A polyacrylamide spike/' (JIsu2sa+) 3 containing a 38 M glycine aqueous solution (hereinafter referred to as liquid A) was prepared. Next, on this glass plate, ampholine (manufactured by LKB, Sweden, pH 4g3.
5-10) containing gel (length 10cm x width 1crn1)
A gel having a thickness of 2 mm) is prepared at the low concentration (4 mm) end of the gradient gel with a gap 4 of 1 mm in between (see FIG. 1(a)). Next, cut a slit 5 in the center of the gel 2, add 10 μL of human serum containing 2% ampholine, and add 0.04 M
Impregnated with a NaOH aqueous solution of 100% and placed a F paper 6 for contacting, and connected to the negative electrode tank 7 containing 0.04M NaOH. The filter paper 8 is connected to a positive electrode bath 9 containing 0.01 M phosphoric acid, and current is applied between both electrodes to separate serum proteins by their isoelectric point difference (see Figure 1(b)).Next Next, remove this liquid junction and hold liquid A in the gap 4 by capillary action.
A liquid junction paper 10 impregnated with liquid A is placed on the gel l, and connected to the negative electrode tank 11. Similarly, the liquid junction door paper 12 impregnated with liquid A is placed at the high concentration end of the gradient gel 3, connected to the positive electrode tank 13, and current is applied between both electrodes to separate the component proteins by their molecular weight. Due to the difference, it separates inside gel 3 (
Above, Figure 1 (C)). Note that during the electrophoretic separation process, the glass substrate 1 is held in a horizontal position.

As described above, using a single glass substrate, isoelectric point separation and
Two-dimensional electrophoretic separation consisting of molecular weight separation was performed. Gel 3 containing separated proteins is handled by handling glass substrate 1 without handling the gel directly, immersing it in an acetic acid-methanol mixture of Komatsu Sheeple-8250, and then immersing this gel (glass substrate) in an acetic acid aqueous solution to remove the protein-containing gel. When the above-mentioned dye present in the portion of the gel that was not present was removed, separated spots of about 100 types of proteins could be detected.

In the present invention, the support for performing the first-dimensional electrophoretic separation is not limited to polyacrylamide gel, and may be other materials such as agarose gel.

That is, agarose gel with a concentration of 1.
．． Two-dimensional migration was performed using the gel as the support in FIG. 1 and Ichiro, and results similar to those obtained when polyacrylamide gel was used as the support 2 were obtained.

As described above, according to the present invention, there is no need to directly handle the two gel-like supports with low mechanical strength at the first and second dimension ports, and it is sufficient to handle the gel together with the hard substrate, so the electrophoretic separation operation is simplified. greatly simplified.

In the present invention, by providing a gap between the first-dimensional and second-dimensional supports for electrophoretic separation, electric lines of force are localized in the first-dimensional supports during first-dimensional electrophoresis, and It also had the effect of increasing the separation power of electrophoresis.

[Brief explanation of the drawing]

FIGS. 1(a), 1(b), and 1(C) are plan views of an electrophoresis apparatus for carrying out the method of the present invention.

Claims (1)

[Claims] 1. In the two-dimensional electrophoretic separation method, a polyacrylamide concentration gradient gel is placed on the substrate, and a polyacrylamide gel or agarose at a constant concentration is placed at a certain gap at the low concentration end of this concentration gradient gel. Using a fixed gel as a support for electrophoretic separation, the first-dimensional electrophoretic separation is carried out within the gel with a constant concentration, and the analyte components separated within the gel with a constant concentration are then separated into the gel with a constant concentration. A simple two-dimensional electrophoresis method characterized by electrophoresis and separation in a concentration gradient gel.