JPH0424658B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is primarily concerned with the separation and analysis of charged colloidal particles such as proteins.

Two-dimensional electrophoresis of proteins utilizes the differences in the physical properties of two types of proteins, and aims to increase the separation ability by combining the two types of electrophoresis. As supports for electrophoresis, polyacrylamide, cellulose acetate membranes, agar (agarose), starch, filter paper, etc. have been put into practical use. Among these, the two-dimensional electrophoresis method combines isoelectric point fractionation using acrylamide in the first dimension and molecular weight fractionation using concentration gradient acrylamide in the second dimension. As technology has improved, it has become possible to perform separation and concentration using isoelectric point difference with an accuracy of about PH1/100, and it has become an excellent protein separation and concentration technology because it concentrates in the direction of migration using a second-dimensional concentration gradient acrylamide gel. be.

Conventionally, the first-dimensional electrophoresis involves polyacrylamide filled in a glass tube, and the second-dimensional electrophoresis involves forming a concentration gradient of acrylamide in a container consisting of a glass plate or an acrylic plate and a spacer, and then polymerizing the plate gel. is used as a support. The first-dimensional gel contains in advance an amphoteric carrier made of various types of polyaminocarboxylic acids or polyamysulfonic acids (for example, LKB's Ampholine, BioRad's Biolite, etc.), and the anode (acidic liquid) and cathode. (Alkaline solution) As a voltage is applied between the two electrodes and migration progresses, a PH gradient is formed within the gel.

Sample proteins loaded on the gel are separated and concentrated at unique isoelectric point positions during the electrophoresis process. When a steady state is reached, this gel is extruded from the glass tube, placed horizontally on top of a two-dimensional flat gel plate, and an electric field is applied between the two electrodes filled with a buffer solution (for example, tris-glycine pH 8.6). The separated components are further separated based on the difference in molecular weight. After the electrophoresis is completed, the container is dismantled and the gel plate is taken out, immersed in a protein staining solution, left to stand, and the background is destained.

Polyacrylamide two-dimensional electrophoresis is an excellent technique for separating and concentrating proteins, but the process is complicated and difficult to handle. The present invention aims to simplify the process without impairing the advantages of this two-dimensional electrophoresis method.

Separation and analysis of proteins using two-dimensional polyacrylamide gel electrophoresis includes (1) isoelectric point fractionation using a rod-shaped polyacrylamide gel, (2) molecular weight fractionation using a concentration gradient acrylamide plate gel, (3) staining of protein components, It consists of four processes: background destaining, (4) computer image processing, and quantification. This process requires manual labor throughout the entire transportation process, and there are many parts that are difficult to automate. The present invention aims to simplify the process without impairing the separation ability of the conventional method, and is mainly concerned with the above steps (1) and (2). In other words, since the concentration gradient gel used in second-dimensional electrophoresis is fixed on one side of the substrate in advance, the gel container can be easily disassembled after electrophoresis, and the gel can be easily handled during the staining and destaining steps. .

After keeping the surface of the substrate (e.g., glass, polyester, etc.) on which the two-dimensional concentration gradient gel is fixed clean, the surface is activated by alkaline treatment (e.g., immersion in 6NaOH solution, washing with water, and drying).
This is then surface treated with a silane coupling agent.
The silane coupling agent (RSiX ₃ ) used here, R
is a compound consisting of an organic residue such as a vinyl group, mercaptopropyl group, methacryloxypropyl group, glycidoxypropyl group, etc., and X is a compound consisting of an organic residue represented by ethoxy, methoxy, acetoxy, etc. Dehydration condensation occurs between the X groups, and bonding occurs between R and the acrylamide unsaturated double bond, thereby immobilizing acrylamide on the substrate.

A fixed concentration gradient plate gel with one side as a substrate is prepared by the following procedure. A plurality of 10 cm square glass plates, each of which has been surface-treated with the above-mentioned silane coupling agent on one side, are prepared as substrates for fixing the gel.
As shown in FIG. 3a, these glass plates 3 are sequentially assembled via spacers so that the silane coupling treated surface 31 of one glass plate and the untreated surface 32 of the other glass plate face each other. Diagram b
As shown in the figure, the gel is vertically inserted into a flat gel manufacturing container 15. A mixed solution of acrylamide monomer, which is the material of the gel, a crosslinking agent, and a polymerization catalyst is poured into the container 15 so that the concentration gradient becomes higher in the lower layers, and in this state, the solution is polymerized by catalytic action to form a polymer. At this time, first, a covalent bond is generated between the basic residue R of the silane coupling agent bonded to the surface 31 of the glass plate by dehydration polymerization and the acrylamide monomer in contact with the surface 31. In other words, the unsaturated double bond at the end of the base residue R and the unsaturated double bond at the end of the acrylamide monomer are
Changes into a series of covalent bonds. In this way, the acrylamide monomer chemically bonded to the surface 31 is further polymerized with other acrylamide monomers to form a polyacrylamide gel, so that each gel plate between the glass plates 3 is chemically bonded to the surface 31 of the glass plate. and fixed. By changing the concentration gradient of the spacer and the mixed solution injected, a single-sided fixed plate gel having an arbitrary thickness and an arbitrary concentration gradient can be obtained.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 In the first dimension of two-dimensional electrophoresis of serum proteins, isoelectric focusing was performed using an acrylamide rod-shaped gel support filled in a glass tube, and in the second dimension, molecular weight determination was performed using a concentration gradient gel with one side fixed to the substrate. It performs fractionation.

For the one-dimensional gel, place an acrylamide mixture (acrylamide monomer 3.8%, NN'-methylenebisacrylamide 0.2%, TEMED 0.028%, ammonium persulfate 0.07%, amphoteric carrier- For example, LKB's Ampholine PH3.5-10; 2% mixed immediately beforehand) is injected to a 9 cm scale and left to polymerize. After contacting the upper end of the gel in the glass tube with the catholyte (for example, 0.04M sodium hydroxide solution) and the lower end with the anolyte (for example, 0.01M phosphoric acid solution),
Place 5μ of serum on the top of the gel and perform electrophoresis at 1000V for about 6 hours. The concentration gradient gel used in the second dimension is a silane-coupled glass plate (100×
A 2 mm thick polyacrylamide plate gel with a concentration gradient of 4 to 21% was chemically bonded and immobilized using the method described above. As shown in FIG. 1, the glass plate 3 on which the flat gel 2 is fixed is placed horizontally on a cooling plate 6. A rod-shaped gel that has undergone one-dimensional migration is placed in a groove prepared in advance on the low-concentration side of the flat gel 2, taking care not to introduce any air bubbles. That is, the container of the two-dimensional electrophoresis apparatus is provided with a cooling plate 6 for horizontally mounting a flat gel 2 fixed on a glass plate 3 and cooling it. Furthermore, this container is equipped with two buffer solution tanks, and the two opposing sides of the flat gel 2 are treated with a tris-glycine buffer solution 7 in these buffer solution tanks via a sponge 5 and a filter paper 4, respectively.
(PH8.6) 7 individually. Each of the two buffer baths is equipped with an electrode 8 for applying an electric field for second-dimensional electrophoresis to the flat gel 2, and horizontal electrophoresis is performed at 500 V for about 3 hours. Note that during this second-dimensional electrophoresis, a cover shown at 9 in FIG. 1 may be placed on top of the flat gel 2. After electrophoresis, the gel is transferred to the staining and destaining process along with the substrate. Through the above procedure, serum is separated into nearly 200 spots, and an image with the same separation power as the conventional method is obtained.

Example 2 This is a combination of isoelectric focusing using a band-shaped acrylamide gel and electrophoresis using a single-sided fixed gel. Place a spacer with a specified thickness in an acrylic container, cover with a glass plate (100 x 100 x 2 mm), and fill the gap with an amphoteric carrier (Ambholine PH3.5-10 manufactured by LKB).
and acrylamide (acrylamide 3.8
%, NN′-methylenebisacrylamide 0.2%,
When 2% ampholine, 0.028% TEMED, and 0.07% ammonium persulfate) are injected and taken out after polymerization, a flat gel adhered to the glass plate is obtained due to the difference in the affinity of the acrylamide polymer gel. Place this horizontally on a cooling plate, and inject 5μ of serum into the hole that was previously made. Both ends of this gel plate are connected to catholyte (0.04M sodium hydroxide solution) and anolyte (0.01M phosphoric acid solution) using filter paper, respectively, and electrophoresis is performed at 1000V for 6 hours. As shown in FIG. 2, the gel 10 cut into strips after the completion of electrophoresis was prepared in the same manner as in Example 1, near the low concentration side of the concentration gradient plate gel 2 with one side immobilized on the substrate 3. The electrode is placed in close contact with or on top of the buffer solution 7, and an electrode is placed on top of the filter paper through a filter paper that is in liquid contact with the buffer solution 7. Further, on the high concentration side of the flat gel 2, a filter paper 4 and an electrode connected to a separate buffer solution are placed, and 500 V is applied between the two electrodes to conduct electrophoresis for 3 hours.

Example 3 Cellulose acetate membrane (e.g.
SeparaxEF) is impregnated with a 2% solution of an amphoteric carrier (for example, Ampholine PH3.5-10 manufactured by LKB), the water is gently wiped off, and then the sample is applied to a predetermined location and isoelectric focusing is performed. After completion of electrophoresis, the cut out tape-shaped membrane was placed on the low concentration side of a single-sided fixed concentration gradient gel, and then subjected to second-dimensional electrophoresis in the same manner as in Example 2.

Example 4 A cellulose acetate membrane is immersed in veronal buffer (PH8.5) in advance, and then the moisture on the surface is wiped off and placed in an electrophoresis apparatus. Serum (1-1.5μ
) to the designated area with a micropipette and run for about 40 minutes with veronal buffer. After the electrophoresis was completed, the cut tape-shaped cellulose acetate membrane was fixed on one side of the concentration gradient gel, and the gel was sandwiched between the incisions made on the low concentration side to prevent air bubbles from entering, and transferred to two-dimensional electrophoresis in the same manner as in Examples 1 and 2. .

As explained above, according to the present invention, by employing an acrylamide concentration gradient gel with one side fixed to the substrate in the second dimension of electrophoresis, the merit of high-performance separation, which is a characteristic of conventional methods, is not lost and the process can be streamlined. It can be simplified. That is,
With conventional flat plate gels simply held on a substrate, electrophoresis is performed with the gel filled in a container consisting of two substrates spaced apart to maintain the shape of the gel.
Next, it was necessary to take out the gel plate inside the container and immerse it in a protein staining solution and a destaining solution in sequence.
In contrast, with the concentration gradient plate gel of the present invention, which has one side immobilized on the substrate by chemical bonding, the gel remains immobilized on the substrate even when it is connected to a buffer solution for electrophoresis. The shape does not change during electrophoresis. Further, after electrophoresis is completed, the gel can be immersed in a staining solution or a destaining solution as it is, and even when immersed in these solutions, the gel remains immobilized on the substrate. Therefore, there is no need to remove the gel from the container or directly handle the removed gel between these processes, making it easy to automate the process. In addition, the shape of the gel is maintained until the final stage, making it possible to accurately analyze the electrophoretic image revealed by staining and destaining.

[Brief explanation of drawings]

Figures 1 and 2 are cross-sectional views of the electrophoresis device;
Figures a and b are a top view and a sketch showing the process of producing a flat gel with one side immobilized in Example. 1... Rod-shaped acrylamide gel after one-dimensional migration, 2... Single-sided fixed concentration gradient gel for two-dimensional migration,
3... Substrate for gel fixation, 4... Filter paper for buffer liquid junction, 5... Sponge, 6... Cooling plate, 7... Buffer solution, 8... Electrode, 9... Cover, 10... One dimension Band-shaped acrylamide gel after electrophoresis.

Claims (1)

[Claims] 1. The migration carrier on which the first-dimensional migration of the sample has been completed is brought into contact with the vicinity of one of the two opposing sides of the flat gel, and the second-dimensional migration is performed within the flat gel.2
In the dimensional electrophoresis device, the flat gel is a concentration gradient polyacrylamide gel chemically bonded and immobilized on a substrate whose one side is subjected to silane coupling treatment, and the two-dimensional electrophoresis device horizontally holds the flat gel. means for holding the two opposing sides of the flat gel or the vicinity thereof with a buffer solution, and two opposing sides of the flat gel.
A two-dimensional electrophoresis device comprising means for applying an electric field for second-dimensional electrophoresis between sides.