JPS60169597A - Method and device for two-dimensional electrophoresis - Google Patents

Abstract

PURPOSE:To enable efficient processing of electrophoresis without replacing supports by positioning the 1st support and the 2nd support to face each other apart at a spacing and executing successively one-dimensional electrophoresis and two-dimensional electrophoresis. CONSTITUTION:The 1st and 2nd supports 1, 2 for one- and two-dimensional electrophoresis are positioned to face each other apart at a spacing 3. A buffer solns. (leading solns.) is poured into the chambers 4a, 4b of a cell 4 and a buffer solns. (final solns.) and buffer solns. (additional solns.) are poured into the chambers 5a, 5b of a cell 5. Both ends of the support 1 are brought into contact with the buffer solns. in the chambers 4a, 5a and electricity is conducted to the electrodes in the chambers 4a, 5a. A sample A such as serum or the like is coated to the cathode end side part on the bottom surface of the support 1 and concd. electrophoresis is executed. The chambers 4b, 5b are then positioned below the support 1 and ordinary electrophoresis is executed. Both cells 4, 5 are retreated and the support 1 is pressed to the 2nd support 2. After constant current is passed from a current conducting terminal 10, the support 1 is detached from the support 2 and a constant current is supplied then the support 2 is subjected to a prescribed treatment and is dried. The characteristic of the sample A is thus judged.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a two-dimensional electrophoresis method and apparatus for completely separating serum and other samples.

Conventionally, as a method for separating various proteins in the composition from serum, a serum solution is applied to the end of a long cellulose acetate membrane support a as shown in Figure 1, and a one-dimensional electrical method is applied to this. , Next, as shown in Figure 2, the entire electrophoresed support a is brought into contact with a support C of a cellulose acetate membrane prepared separately, and the electrophoresis direction of the support a is perpendicular to the support C.
A two-dimensional electrophoresis method is known in which dimensional electrophoresis is performed. According to this two-dimensional electrophoresis method, it is possible to separate the composition separated wave-like on the support by one-dimensional electrophoresis in more detail for each wave, and the support CF can be fixed, dyed, destained, and dried. It has the advantage that by reading all the patterns that appear, it is possible to analyze all types of proteins in serum that cannot be seen by one-dimensional electrophoresis.

One-dimensional electrophoresis, which is performed as a pre-step to two-dimensional electrophoresis, is condensation electrophoresis, in which the sample on support a is once concentrated, and the composition of the sample, which has different mobilities under constant pH conditions, is compatible. It is necessary to sequentially perform so-called ordinary electrophoresis, which separates the sample, and by performing the concentration electrophoresis, it becomes possible to accurately separate minute amounts of components in a small sample into one-dimensional separation.

In the process of performing all of these two-dimensional electrophoresis, the slow spinal fluid used for concentrated electrophoresis and the buffer solution used for normal electrophoresis are different types of liquids, and as the process progresses, the In addition to the need for replacement, it is also necessary to transfer the support A after 1D electrophoresis to a support C prepared separately by hand for 2D electrophoresis, so that the process can be carried out efficiently. However, there are drawbacks such as the fact that foreign matter adhering during transfer may disrupt the analysis.

The present invention aims to eliminate all of these drawbacks and inconveniences, and its first invention is a one-dimensional city.

A first support for gas phoresis and a second support for two-dimensional electrophoresis are opposed to each other with a gap between them, and a concentrated electrophoresis buffer and a normal electrophoresis are placed on the sides of the first support. A buffer solution tank for electrophoresis is arranged, and at least one of the first support and the electrophoresis is completely moved, and the first support is sequentially exposed to each buffer solution for concentration electrophoresis and normal electrophoresis. The second invention relates to a method characterized in that one-dimensional electrophoresis is performed by bringing the first support into contact with an opposing second support, and then two-dimensional electrophoresis is performed by bringing the first support into contact with an opposing second support. A first cooling tank having a first support for one-dimensional electrophoresis attached to its outer surface and a second cooling tank having a second support for two-dimensional electrophoresis attached to its outer surface are connected so that both supports are spaced apart from each other. At least one of the two cooling tanks is provided so as to be movable so that both supports are in contact with each other, and a concentrated electrophoresis buffer solution and a +lt! A retentate tank containing a conventional electrophoresis buffer in all the compartments is provided, and at least one of the first cooling tank and the buffer tank is moved so that the first support sequentially contacts each of the buffer solutions. It concerns a device that can be freely configured. ) An embodiment of the first invention of the present invention will be described with reference to FIG. 5 of the drawings. (2) shows a second support used for two-dimensional electrophoresis, which is provided with a membrane surface of cellulose acetate and is arranged to face the first support (1) with a gap (3) between the two supports. , the second support (2) has a width equal to that of the first support (1).
Although it is somewhat shorter than the length of the first support (IJ
It has a sheet shape that is a little longer than that of the original. Both supports (1) t2
) are arranged perpendicular to each other, and the upper surface of the first support (1) and the lower surface of the second support (2) are each held by a rigid member.

(4) (51 indicates a buffer tank located on both sides of the first support (1) and having an opening above; both 1'i)
(4) (51 is provided movably in synchronization with the direction orthogonal to the first support (1). Both +1 (4)
It is also possible to fix (5) and move the first support (1) in its width direction instead.

Various types (4) (51 is a buffer solution for concentrated electrophoresis and a buffer solution for normal electrophoresis. 2 compartments each so that they can be accommodated.
It is divided into chambers (4a), (4b), (5a), and (5b), and each chamber is provided with an electrode (6), a first support (1), and a second support via a binding liquid supply paper (8). A supporting mound (force) is provided above (2). (9) is glass fiber for electrodes placed on both ends of the second support (2), +l1
l) Glass fiber for electrode (9) used in two-dimensional electrophoresis
It is a knife-shaped energizing terminal that moves up and down to make contact with the terminal.

A specific example of the method of the present invention is as follows. As the first support (1), a cellulose acetate membrane with a relatively high electroosmotic pressure of 1, for example, a commercially available product under the trade name Titan ■, is selected. A commercially available product with different properties and almost no electro-osmotic pressure (trade name: Separax E F cellulose acetate)
1 model'eF2 support (2) was selected as the first support (1)'r 62 mmol/1 Tris-HCl, P H
The second support f2)'5 is mixed with the liquid of 6 and 7.
F 113.5 to 10.0 5% Amph line,
The i
≦i. It n in chambers (4a) (4b) of tank (4)
i+ notation fc, 6 2 mmol/l tri
Pour liquid (lead liquid) into the tank to make it the anode.
50 m to Ina (5)'s room (5a) while attending the errands of Ton.
mal/l arginine-barium hydroxide PH11,
Pour the buffer solution (final solution) in Step 7, and prepare the entire cathode for bending.

In addition, 100 m mol was added to the other chamber (5b) of the tank (5).
/l Tris-IJ acid, pH 7.5 buffer solution (molding solution) is poured, and preparations are made for energization to use the electrode (6) as a cathode.

When the above preparations are completed, first move the first support (1) down so that each end is in contact with the buffer solution in the chambers (4a) and (5a), and then attach the first support (1) to the electrodes in each chamber (4a) and (5a). Preliminarily apply a constant voltage of 200 V DC for 5 minutes. Thereafter, a sample such as serum is applied to the cathode side end of the lower surface of the first support (1), and the amphoteric carrier component in the sample A is transferred to the chamber (4a ) with the leading ion in the buffer of the chamber (5
It is sandwiched between the final ions in the buffer solution in a) and migrates from the cathode side to the anode side. When the electroosmotic pressure acting in the opposite direction is balanced, the migration stops and concentration electrophoresis begins. Finish and dry.

Next, move the tank (41 (51) so that the chambers (4b) (5b) of the tank (41 (51) are located below the first support (1), and open both ends of the first support (1). Each room (4b) (
5b) while contacting the loose sleeve liquid and applying 0.4 to the electrode.
Regular electrophoresis is carried out by applying a constant current of mA for about 45 minutes.

This completes the one-dimensional electrophoresis.

For two-dimensional electrophoresis, both vessels (41 (51 is the first support (1)
The second support (2) is removed from the bottom of the support (1).
], a constant current of imA is applied from the current terminal (10) for about 1.5 hours, and then the voltage is increased to aoov, causing the entire first support (1) to separate from the second support (2) for about a
A constant voltage of oov is applied for about 1.5 hours and the process is completed.

The second support (2) was then treated with a fixing solution, a staining solution, and a destaining section, and when dried, the composition of sample A was developed.
A dimensional development image appears, and the 1m size of the characteristics of sample A becomes a DJ function.

Using this method, move the first and second supports (11 (2) up and down slightly without touching them by hand) until the tank (4) (
5) Each stage of electrophoresis (f) up to two-dimensional electrophoresis can be performed continuously by simply moving the electrophoresis system, thereby eliminating the possibility of analysis errors due to errors in the electrophoresis procedure.

An embodiment of the second invention of the present invention is as shown in FIGS. 4 and 5, in which the first support (1) is placed in a closed box-shaped first cooling tank provided with a cooling liquid circulation port αυθat. A second cooling W in the form of a dense 12J box is attached to the mounting plate 03) below the outer surface of the
Attach it above the outer surface of 111'3. The first cooling (soda (1
2+ and the second cooling tank t151 are the first and second supports +1+
+2) are arranged so that If41 lψA (3) k exist and face each other. In addition, the first cooling tank 0z has a support shaft t16) formed by extending both sides of the tank (4) with full valves and a left and right movement position.
The edge (17) of 1(5) shall be supported by a stake, and the second cooling tank 09 shall be slidably provided on the bottom plate Cυ. The end of each shaft (16) is connected to the left and right tanks (41 (5)
The first cooling tank 02) entered into a long hole (20a) formed in a fixed side plate 4 on the outside of the first cooling tank 02), so that only vertical movement was allowed in the first cooling tank 02). ul is a connecting member for moving both tanks (4) (51) in synchronism. 4a) A buffer solution for concentrated electrophoresis is injected into (5a), and a buffer solution for normal electrophoresis is injected into chambers (4b) and (5b).Also, each chamber has an electrode consisting of a self-contained wire. (
6) is provided, and a knife-shaped current-carrying terminal 00) is provided so as to be movable up and down relative to the electrode glass fibers (9) at both ends of the second support (2), which is similar to that shown in the third figure. . On each edge α7) (+8) of both tanks (4) and (5), there are two cam grooves (17a) (17a) (
17b) (18a) (18b) and cam grooves (17a) (18c) with large lifts are formed with a gap between them, and when both tanks (4) (51'e) are moved, the first cooling tank Concentration electrophoresis, normal electrophoresis, and two-dimensional electrophoresis were performed by ascending and descending sequentially along the groove. (221
is a buffer spring interposed between the first and second cooling tanks Q2) (15).

To illustrate its operation, the first support (1,) is grooved with a buffer solution as indicated in Figure 4, and the second support (2) is wetted with an amphorain solution; Cooling tank (
121 and the second cooling tank (15).

Next, when the tank (41 (51) is moved, the support shaft (16) falls into the cam groove (17al (18a), and the first cooling tank circle also descends, and both ends of the first support (1) is placed on the buffer supply paper (8) on the mound (7) of the chamber (4a) (5a) which is 2" filled with liquid. Here, the chamber (4a)
The electrode (5a) (after passing 0111 to 61, place it under the first support (1) at 81↓5 in the same way as in the case shown in Fig.
When the first support (1) is cooled, concentration 1E aerophoresis is performed. After that tank (4) (
5), the first cooling tank (I2) rises, but descends again along the cam groove (17b) (17b),
The first support (1) has mounds (7) of chambers (4b) (5b).
), so if you apply electricity to that electrode (6), 11
Can perform normal electrical procedures. Furthermore, the tank (41 (5)
If you move it to 1, the cam 1174 (17c) has a large lift.
) (18c), the first support (1) comes into contact with the second support (2), and at this time the current-carrying terminal (
10) If electricity is applied from (10), on the second support (2),
Two-dimensional electrophoresis can be performed while cooling.

In this second invention, the first and second supports (1) (2
1 One of the first and second cooling tanks A21 (ls attached to the entire outer surface) can be fully moved to make the support body (1) (2+ fully contactable, and the 1 cooling tank O and the holder (4) (51 gold) Since it is relatively movable, it is possible to move rJ at each stage of two-dimensional electrophoresis.
! The process can be carried out sequentially while cooling the support (11(2+).

In this way, according to the present invention, by slightly moving either the first support or the second support that faces each other, the first support or the tank (if either one) can be moved in two-dimensional electrophoresis. Multi-level electrophoresis can be performed completely sequentially, eliminating the need to transfer supports, allowing for efficient electrophoresis processing.Furthermore, according to the second invention, supports are provided in opposing cooling tanks. , move the cooling tank and support at the same time!1
Since the melt is allowed to stand on its own surface, it has the effect of being able to perform aerophoresis while being cooled during transportation.

[Brief explanation of drawings]

Fig. 1 is a perspective view of a support for one-dimensional (1) L pneumophoresis; Fig. 2 is a perspective view of a 642-dimensional redundant electrophoresis method;
FIG. 4 is a perspective view of the embodiment y0 of the invention, FIG. 4 is a perspective view of the practical example of the second defense of the invention, and FIG. 5 is a side view taken along the line V-V. (1)...First support (2)...T:82 support (3)...Interval (4) (5)...tank (4at
(4b) (5a) (5b) -ku]l straw (1”1
f15)...2 people outside the cooling tank

Claims (1)

[Claims] 1. A first support for one-dimensional electrophoresis and a second support for two-dimensional electrophoresis are placed opposite to each other with a gap between them, and a buffer solution for concentrated electrophoresis is placed on the side of the first support. and a tank containing a buffer solution for normal electrophoresis to move at least one of the first support and the nuclide relative to each other. A two-dimensional electrophoresis method characterized in that one-dimensional five-particle electrophoresis is performed by sequentially contacting the first support and two-dimensional electrophoresis is performed by bringing the first support into contact with an opposing second support. - A first cooling tank with a first support for two-dimensional electrophoresis attached to the outer surface and a second cooling tank attached to the entire outer surface of the second support for two-dimensional electrophoresis are mutually connected to each other. The cooling tanks are provided so as to face each other with a gap therebetween, and at least one of the cooling tanks is provided so as to be movable at will so that both the supporting bodies are in contact with each other.
A buffer solution tank containing a concentrated electrophoresis buffer solution and a normal electrophoresis buffer solution in a compartment is provided on both sides of the cooling tank, and at least one of the first cooling tank and the buffer solution tank is provided with a first support. A two-dimensional electrophoresis device configured to be movable so that the body sequentially contacts each of the buffer solutions.