JPS6139617B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gradient gel for electrophoresis and a method for producing the same.

Conventionally, electrically charged substances such as albumin, globulin, other proteins, and nucleic acids contained in serum, cerebrospinal fluid, urine, etc., are separated and analyzed by electrophoresis on an electrophoresis support, generally known as electrophoresis. There are known analytical methods for Recently, electrophoresis methods using a gel produced by polymerizing acrylamide in the presence of a cross-linking agent as a support for migration, or a method in which the amount of polyacrylamide is gradually increased in the direction of migration when polymerizing acrylamide, have been developed. A method has been reported in which a manufactured gradient gel is used as a support for electrophoresis.

When using a gradient gel as described above, the migration of substances with specific molecular forces stops at a specific concentration of polyacrylamide, so it is thought that the separation of substances such as proteins is due to their molecular weights. It is being This property is considered to be the same effect as the so-called molecular sieve effect conventionally known in column chromatography technology.

When using a gradient gel to identify an unknown substance, the known substance is usually added to the sample,
A method has been adopted in which the unknown substance is identified by comparing the stopping position of the known substance and the unknown substance.

In this method, the unknown substance is identified based on its relative position to the electrophoretic position of the known substance, so it is always necessary to add the known substance.

On the other hand, when producing the gradient gel used,
It is particularly difficult to maintain a constant gradual increase or decrease (gradient) in gel concentration, and in practice, even within a single gel, changes in gel concentration may be rapid or gradual in some parts. This is the current situation. Therefore, it is particularly difficult to measure the molecular weight of an unknown substance from the electrophoresis position of a known substance.

The present inventor conducted research and completed the present invention with the aim of creating a gradient gel for electrophoresis that does not require the addition of known substances and can easily detect the gel concentration at a specific position.

That is, the gist of the present invention is as follows: (1) It consists of a polymer molded product obtained by polymerizing a mixed aqueous solution containing a monomer, a crosslinking agent, a polymerization initiator, and a dye in a molding machine, and in the polymer molded product, The polymer has a concentration gradient in the direction of electrophoresis, and the dye has a concentration distribution that is the same as or opposite to the concentration gradient of the polymer, and each position of the dye concentration corresponding to the stop position of the standard substance is marked. Gradient gel for electrophoresis (2) Two types of aqueous solutions containing monomers, crosslinking agents, and polymerization initiators and having different concentrations are mixed while gradually changing the mixing ratio. In the method for producing a gradient gel for electrophoresis having a concentration gradient of the polymer in the direction of electrophoresis, the above-mentioned two types By adding a dye to one side of the aqueous solution, the content of the dye in the gel is gradually decreased or increased in the electrophoresis direction, and then the electrophoresis stop position of the standard substance measured separately is correlated with the dye concentration, and the stop position is marked. A method for producing a gradient gel for electrophoresis, characterized in that:

The apparatus for preparing a gradient gel for electrophoresis of the present invention will be explained with reference to FIG. It consists of a container 4 filled with a low concentration aqueous solution 3 containing a polymer, a crosslinking agent, a polymerization initiator, a dye, etc., a pump 9, and a molding device 11. The container 4 and the pump 9, and the pump 9 and the former 11 are connected by a conduit 7 having a conduit 8, respectively.
and 10, which is a gradient gel preparation device for electrophoresis.

In this device, the part consisting of the containers 2, 4 and the pump 9 is shown as a linear device that changes the concentration linearly, but it is also a device that is generally called a gradient device that changes the concentration of the liquid continuously.
For example, in addition to the above-mentioned linear type, any type such as a convex type or a concave type can be used.

The pump 9 in Fig. 1 is a so-called peristaltic pump that pumps out the solution in the tube by sliding the tube from the outside, but it is a pump that continuously moves a droplet-sized amount of the solution. Any of these can be used.

The molding device 11 in FIG. 1 is shown as a square molding device, but it may be of any shape, such as a cylinder or a plate, depending on the shape of the gradient gel required. Bye.

Now, an example of gradient gel preparation when a dye is added to a highly concentrated solution will be described below.

A highly concentrated aqueous solution containing monomers, crosslinking agents, polymerization initiators, polymerization accelerators, etc. dissolved in an appropriate buffer solution is placed in container 2.
and charging the low concentration aqueous solution into the container 4,
The stirrer 5 is rotated and the pump 9 is activated and at the same time the valve 6 is opened. A low concentration solution is introduced from the lower part of the molding machine 11 from the container 4 by the pump 9, and at the same time, a high concentration solution is introduced from the container 1 by gravity into the container 4.
The liquid height in both containers 2 and 4 is always kept equal, and the high concentration solution 2 is added to the low concentration solution 3.
enters through the valve 6, the concentration of the low concentration solution 3 gradually increases, and the dye concentration of the low concentration solution 3 gradually increases due to the dye added to the high concentration solution. Therefore, the dye concentration and monomer concentration of the solution fed from the lower part of the molding machine 11 by the pump 9 gradually increase, so that the molding machine 1
In the solution in 1, the monomer concentration increases continuously from the top to the bottom, and the dye concentration increases along with this concentration.

The mixed solution thus introduced into the molding device 11 is allowed to stand for a while to complete polymerization, harden into a gel-like (agar-like) state, and is molded into the shape of the molding device. By measuring the dye concentration of the resulting gel using a suitable absorbance meter, the concentration of the polymer produced from the above monomer and crosslinking agent (hereinafter referred to as gel concentration) can be determined in proportion to the dye concentration. be able to. Next, mark the gel concentration determined from the dye concentration on a gradient gel or gradient gel container, use a gradient gel in which the type and concentration of monomers, crosslinkers, etc. are constant, and use standard substances (for example, molecular weight A known protein) is electrophoresed, and the position where a substance with a specific molecular weight stops migrating is detected. By marking the same monomer concentration on the same gradient gel in advance (marking), in other words, by correlating the dye concentration and the migration position of a specific substance, the migration position of an unknown substance can be determined. From this, the molecular weight of the substance can be known and the substance can be identified.

Examples of monomers used in the present invention include acrylamide and methacrylamide.

As a crosslinking agent, N,N'-methylenebisacrylamide, N,N'-propylenebisacrylamide, diacrylamide dimethyl ether,
1,2-diacrylamide ethylene glycol,
Examples include ethylene ureabisacrylamide and the like.

The concentration of crosslinking agent is approximately 2 to 30% of the monomer.
% by weight is used, preferably about 3-5% by weight.

Any dye that is soluble in water can generally be used, but for example, a dye that has an electric charge can be particularly effectively used because it can be removed by electrophoresis.

Examples of the dye used in the present invention include Brome Phenol Blue and Brome Phenol Red. The amount of dye to be added may be any concentration as long as the change in dye concentration and absorbance are linear, but if Brome Phenol Blue is used, a solution containing monomers etc.
Approximately 2 to 4 mg per 100 ml is suitable.

Monomers, crosslinking agents, polymerization initiators, etc. are usually dissolved in a buffer solution, but any material with ionic strength that does not cause PH changes during electrophoresis can be used. , a buffer solution of trishydroxyaminomethane and boric acid is preferably used.

The gradient gel for electrophoresis of the present invention is prepared using the apparatus and method described above, but it is easy to use because the gel concentration or the migration position of a substance having a specific molecular weight can be marked in advance. , it is possible to identify unknown substances without adding standard substances.

Next, the present invention will be explained with reference to Examples, but the present invention is not limited to the following Examples.

Example 1 Preparation of buffer solution 10.75 g of trishydroxyaminomethane, 5.04 g of boric acid, and 0.93 g of ethylenediaminetetraacetic acid disodium salt were dissolved in water to make 1 (hereinafter referred to as tris-borate buffer solution). PH of this buffer solution
was 8.3.

Preparation of highly concentrated solution Acrylamide 172.8g, N,N'-methylenebisacrylamide 7.2g, tetramethylethylenediamine 0.3ml, bromophenol blue 9mg
and 0.3 g of ammonium persulfate were dissolved in the above Tris-borate buffer solution to make 500 ml, and thoroughly degassed under reduced pressure.

Preparation of low concentration solution: Dissolve 23.04 g of acrylamide, 0.96 g of N,N'-methylenebisacrylamide, 0.3 ml of tetramethylethylenediamine, and 0.3 g of ammonium persulfate in a Tris-borate buffer solution to make 500 ml, and thoroughly degas it under reduced pressure. .

Preparation of Gradient Gel A polyacrylamide gradient gel is prepared using the apparatus shown in FIG. First, fill the conduit 10 with 10% ethyl alcohol, put the high concentration solution in container 2 and the low concentration solution in container 4, rotate the stirrer 5, then operate the pump 9, and immediately turn off the valve. Open 6. The molding device 11 has an inner diameter of 82 mm in length, 73 mm in width, and 3 mm in thickness.
Six glass containers each having a small hole through which an acrylamide solution could be injected were connected to each other so that a gradient gel could be formed at one time.

The pump speed was approximately 25 ml/min.

When the alcohol initially introduced into the conduit 10 rose to the upper end of the molding machine, the pump was stopped and discharged for 20 minutes to polymerize acrylamide.

Reference photo 1 is a polyacrylamide gradient gel prepared in this example.

Next, the absorption of bromophenol blue in the acrylamide gradient gel was measured using a densitometer at a wavelength of 610 nm, and FIG. 2 was obtained.

On the other hand, similar absorbance was measured using a similar gel obtained only from a low concentration solution containing dye at the same addition rate for a high concentration solution and acrylamide, and was used as a standard.

In FIG. 2, the horizontal axis is the length of the gradient gel and the vertical axis is the absorbance (-logT).

It is clear from Figure 2 that the change in acrylamide concentration (already polymerized at this time) increases linearly, and by proportionally distributing the dye concentration and gel concentration, the gel concentration at a specific position can be adjusted. It can be determined by calculation.

The gradient gel prepared in this example had a linear gradient of gel concentration from 4.8 to 30%.

The gel concentration was then marked on the edge of the gradient gel. The dye used in this gradient gel is
The sample protein can be completely removed by electrophoresis before applying it.

Reference photo 2 is a photo of a standard protein electrophoresed using the polyacrylamide gradient gel prepared in this example, and (1) is a photo of a standard protein with a molecular weight of 45,000.
OV-albumin, (2) is a bobbin with a molecular weight of 67,000.
Cerium albumin, (3) is catalase with a molecular weight of 240,000, and (4) is ferritin with a molecular weight of 540,000.
All of the above standard proteins were products of Boehringer Mannheim. Staining was done with 0.1% Coomasie blue (Coomasie blue).
The gradient gel was immersed in Blue) R250.

The protein molecular weight and electrophoresis position were determined from this electrophoresis position and FIG. 2, and the results are shown in FIG.

In this way, the acrylamide concentration and the position where a protein of a specific molecular weight stops migrating are detected in advance, and a mark is placed in advance on a polyacrylamide gradient gel prepared in the same way, and the position where a protein substance of known molecular weight stops migrating is detected in advance. By correlating this with the concentration position, the molecular weight of the protein can be determined from the migration position of the unknown protein.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of an apparatus for preparing a gradient gel for electrophoresis, Figure 2 is an absorbance curve measured by a densitometer of the gradient gel for electrophoresis prepared in Example 1, and Figure 3 is a standard diagram. This is a straight line indicating the molecular weight of the protein, the electrophoresis position, and the monomer concentration in the gradient gel. 1... High concentration solution, 2... Container, 3... Low concentration solution, 4... Container, 6... Valve, 9... Pump, 1
1... Forming device In Fig. 2, the vertical axis shows the absorbance, and the horizontal axis shows the length of the gradient gel. 201...Absorbance curve, 202...Length of gradient gel (relative value), 203...Absorbance of standard gel obtained from high concentration solution, 204...Absorbance of standard gel obtained from low concentration solution, 205 …
...Difference in standard gel absorbance obtained from high and low concentration solutions In Figure 3, the vertical axis shows the standard protein molecular weight, and the horizontal axis shows the gel concentration (%). 301...OV-albumin, 302...bobbin cerium albumin, 303...catalase, 304...ferritin.

Claims (1)

[Scope of Claims] 1 Consists of a polymer molded product obtained by polymerizing a mixed aqueous solution containing a monomer, a crosslinking agent, a polymerization initiator, and a dye in a molding machine, in which the polymer is It has a concentration gradient in the direction of electrophoresis, and the dye has a concentration distribution that is the same as or opposite to the concentration gradient of the polymer, and each position of the dye concentration corresponding to the stop position of the standard substance is marked. Gradient gel for electrophoresis. 2. Introducing two types of aqueous solutions containing monomers, crosslinking agents, and polymerization initiators and having different concentrations into a molding machine while gradually changing the mixing ratio;
In a method for producing a gradient gel for electrophoresis having a polymer concentration gradient in the direction of electrophoresis, the polymerization of a monomer and a crosslinking agent is completed in the molding machine,
By incorporating a dye into one of the two types of aqueous solutions mentioned above, the content of the dye in the gel is gradually decreased or increased in the direction of electrophoresis, and then the stop position is determined by correlating the dye concentration with the separately measured stop position of the standard substance. 1. A method for producing a gradient gel for electrophoresis, the method comprising marking a gradient gel for electrophoresis.