JPH01260356A - Sample coating for electrophoresis - Google Patents

Abstract

PURPOSE:To facilitate a coating operation while increasing a coating level of a sample, by coating a sheet-shaped electrophoretic film with the s ample applying a voltage between the sample body and the sheet-shaped electrophoretic film to move the sample easily to the sheet-shaped electrophoretic film. CONSTITUTION:A sample coating jig 11 is formed in a slender U shape with an interval 15, a slot 16 is provided at a curved surface part of a bottom and two windows 17 are arranged as opposed to each other on the top of the slot 16. A sheet-shaped electrophoretic film 12 is formed on the top of a smooth glass plate 19. To coat the sheet-shaped electrophoretic film with the sample, first, the tip of the sample coating jig 11 is immersed into a container where the sample 4 is housed to make the sample 4 inter into the interval 15 and after it is held by a specified amount, a cathode side terminal 13 of a DC power source is connected to a terminal 18 for electric connection while an anode side terminal 14 to the sheet-shaped electrophoretic film 12. Thus, a voltage of 50V is applied and the tip of the jig 11 abuts the sheet-shaped electrophoretic film 12 to coat the electrophoretic film 12 with the sample 4.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for applying protein or nucleic acid of a sample to a sheet-like electrophoretic membrane in electrophoresis of biological components.

[Conventional technology]

Electrophoresis is extremely important in the field of genetic engineering as a means for separating biological components such as various proteins and nucleic acids (DNA, RNA, etc.).

In this electrophoresis method, a sample is applied to an electrophoresis medium, and then an electric field is applied to the electrophoresis medium to separate desired biological components.

Electrophoresis media used in electrophoresis include cellulose acetate membranes, agarose gels, polyacrylamide gels, filter paper, and the like. Among these electrophoretic media, polyacrylamide gels and agarose gels are preferred from the standpoint of precise separation analysis, and polyacrylamide gels are most commonly used because they allow separation using molecular sieves.

By the way, since the gel membrane of polyacrylamide gel and agarose contains a large amount of water, it is difficult to apply the sample by bringing the sample application jig holding the sample into contact with the gel membrane. However, only a small amount of sample could be coated, and there was a problem with sensitivity.

Therefore, conventionally, in order to sufficiently coat a sample specimen on a sheet-like electrophoretic membrane such as polyacrylamide gel, as shown in FIG. A well 2 for injection was formed, and a sample 4 was injected into the well 2 from above using a microsyringe 3 or the like.

In addition, the code|symbol 5 is a spacer, the code|symbol 6 is a flat glass, and the code|symbol 7 is a notched glass.

[Problem to be solved by the invention]

Inject sample 4 into well 2 using the conventional microsyringe 3 described above.
In this method, the tip of the microsyringe 3 had to be positioned approximately at the center of the small well 2, which was extremely troublesome and difficult to operate.

In addition, because it is injected into the well 2 for coating, it is necessary to use a vertical electrophoresis method in which the sheet-like electrophoretic membrane 1 is stood upright. I couldn't do it.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for applying a sample in electrophoresis, which solves the above-mentioned problems and allows a sufficient amount of sample to be applied with extremely simple operations.

[Means to solve the problem]

In order to achieve the above object, the present invention applies a sample while applying a voltage between a sample specimen and a sheet-like electrophoretic membrane.

That is, the sample application method in electrophoresis of the present invention is as follows:
In a method of applying a sample to a sheet-like electrophoretic membrane having a hydrous gel, the sample is held in an electrically conductive sample application jig, and the sample application jig is used as a cathode (an electrical connection terminal on the low potential side of a DC power source). The sheet-shaped electrophoretic membrane is then connected to an anode (an electrical connection terminal on the high potential side of a DC power supply), and the sample is applied to the sheet-shaped electrophoretic membrane while applying a voltage between the sample and the sheet-shaped electrophoretic membrane. It is structured with this feature in mind.

The sample application jig for holding the sample can be formed in various structures as long as it can temporarily hold the sample and bring it into contact with the sheet-shaped electrophoretic membrane. For example, it may be held in a space formed by substantially parallel plates, or it may be provided with a microporous material and held within the pores.

Further, although the sample application jig is electrically conductive, it may have a structure that allows electricity to be applied to the sample held, and the entire jig does not need to be made of an electrically conductive material. For example, it may be made of plastic, and the sample holding portion may be provided with an electrically conductive material. The electrically conductive substance is not particularly limited, but metals such as stainless steel and platinum, carbon, etc. are preferable.

The type of voltage to be applied may be any method that causes the sample to substantially move to the electrophoretic membrane (e.g., direct current voltage, pulse voltage, etc., and preferably direct current voltage.The applied voltage is
It is used in a range of about 0.1 v to about 1000 v, and the voltage application time is substantially the time during which the sample moves, and is about 0.1 v to about 1000 v.
A range of 1 to about 10 minutes is used.

Although known electrophoretic media can be used for the sheet-like electrophoretic membrane, polyacrylamide gel, agarose gel, etc. are particularly suitable. These may be supported by a glass plate or a film such as polyethylene terephthalate. Further, there is no particular restriction on the composition of the buffer solution included in the gel medium, and any known buffer solution can be used.

As the electrophoresis method, a horizontal electrophoresis method, a bridge electrophoresis method, etc. can be used.

The sample application method of the present invention can be applied not only to sheet gel electrophoresis membranes but also to gel electrophoresis media of other shapes, such as columns (
It can also be applied as a method for injecting a sample into a columnar gel electrophoresis medium.

[Effect]

In the sample application method for electrophoresis of the present invention, since a voltage is applied between the sample and the sheet-shaped electrophoretic membrane, the sample easily transfers to the sheet-shaped electrophoretic membrane, and the amount of sample applied can be increased. In addition, when the sample application jig is brought into contact with the sheet-shaped electrophoretic membrane, the sample application jig is placed from above the sheet-shaped electrophoretic membrane arranged horizontally, so the degree of freedom in the operation space of the sample application member is increased. It is extremely high and can facilitate the coating operation.

〔Example〕

An embodiment of the sample application method for electrophoresis of the present invention will be described based on FIGS. 1 to 3.

FIG. 1 is a perspective view showing a sample application method, FIG. 2 is a sectional view of the same, and FIG. 3 is a perspective view of a coating jig used in the same.

In FIG. 1, the reference numeral 11 is a sample application jig, and the reference numeral 12 is a sheet-like electrophoretic membrane. It is connected to the anode side terminal 14.

As shown in FIG. 3, the sample application jig 11 is made of stainless steel and is formed into an elongated r U J shape with a slight gap 15, and has an elongated hole 16 bored in the curved surface at the bottom. , two windows 17.17 are provided at the top of the elongated hole 15.
are bored facing each other. Further, an electrical connection terminal 18 is provided on the upper end surface, and this electrical connection terminal 18 is connected to the cathode side terminal 13.

The sample application jig 11 has a width of about 3 mm to about 10 mm, and a gap 15 of about 2001 m to about 1000 mm! m,
When the sample 4 is held up to the position indicated by the two-dot chain line in the figure, the amount of the sample 4 held is approximately 1 Ill to 10 m.

Further, the sheet-like electrophoretic film 12 is formed on the upper surface of a smooth glass plate 19, and spacers 2 are provided on both sides thereof.
0.20 is provided.

In order to apply a sample to a sheet-like electrophoretic membrane with the above configuration, first, the tip of the sample application jig 11 (2 in FIG.
(the part below the dotted chain line) is immersed in the container containing sample 4,
The sample 4 enters the gap 15 from the long hole 16, the window 17, and the side surface of the gap 15, and is held in the gap 15 by a predetermined amount.

Next, as shown in FIG. 2, the cathode side terminal 13 of the DC power source is connected to the electrical connection terminal 18, the anode side terminal 14 of the DC power source is connected to the sheet-like electrophoretic membrane 12, and a voltage of 50 V is applied. At the same time, the tip of the sample application jig 11 is brought into contact with a predetermined portion of the sheet-like electrophoretic membrane 12 from above. Then,
The sample 4 held in the gap 15 is attached to the sheet-like electrophoretic membrane 1
In addition to the water absorbing property of 2, it is reliably applied to the sheet-like electrophoretic membrane 12 due to the influence of the voltage.

4 to 7 are perspective views showing other examples of the sample application jig.

The sample application jig 11 shown in FIG. 4 has the same dimensions and shape as the sample application jig 11 shown in FIG.
A platinum wire 21 is stretched from the hole 8 and stretched into the elongated hole 16.

The sample application jig 11 shown in FIG.
2 and a spacer 23, it has a rectangular shape.

The sample application jig 11 shown in FIG. 6 is formed by adhering a stainless steel plate 22 and a plastic plate 24 with spacers 23 and 23 interposed therebetween, and the electrical connection terminal 18 is fixed to the stainless steel plate 22.

The sample application jig 11 shown in FIG. 7 is formed by bonding two plastic plates 24 and 24 at the bottom with a microporous conductive material 25 interposed therebetween. The connection terminal 18 is connected with a platinum wire 21.

In this sample application jig 11, the sample is not held in the gap 15 but in the micropores of the microporous conductive material 25.

FIG. 8 is a front view of a multiple sample application jig 26 in which a plurality of sample application jigs 11 are integrally provided.

In this multiple sample application jig 26, a plurality of sample application jigs 11 are fixed to a mounting plate 27, and these sample application jigs 11...
- Eleven electrical connection terminals 18...18 are connected together. With this multiple sample application jig 26, all the samples 4 can be applied to the sheet-shaped electrophoretic membrane 12 in one operation.

Next, experimental results comparing an example of the present invention and a conventional example will be explained.

Example 1 As shown in FIGS. 1 to 3, a sheet-shaped electrophoretic membrane 1
2 is placed horizontally, and the sample application jig 11 holding approximately 2Ill of the sample is brought into contact with the sheet-like electrophoretic membrane 12 from above, and the sample 4 is applied to the sheet-like electrophoretic membrane 12 while applying a DC voltage of 50 V. I moved it to .

Conventional Example I The experiment was carried out using a device similar to that of the embodiment, but by simply bringing the sample application jig 11 into contact with the sheet-shaped electrophoretic membrane 12 without applying a DC voltage.

Conventional Example H As shown in FIG. 9, a sheet-like electrophoretic membrane 1 is vertically arranged and a microsyringe 3 is injected with approximately 2t11 of sample.
was inserted into well 2 from above, and then sample 4 was discharged.

In the above, the sheet-shaped electrophoretic membrane contains acrylamide (11,87 g), N,N'-methylenebisacrylamide (0,63 g), SDS (0,10 g),
Tris(hydroxymethyl)aminomethane (0,303
g), glycine (1.44 g) in addition to water 100 d
After that, 5 ammonium persulfate was added as a polymerization initiator.
An acrylamide gel composition solution (gel forming solution) was prepared by adding wt% aqueous solution (3.9d) and N,N,N',N'-tetramethylethylenediamine 33IJ1, and two flat glass plates with smooth surfaces were prepared. A glass plate cell having a thickness of 400 tm was prepared using a spacer with a thickness of 400 tm, and the above gel forming solution was injected into the cell and gelled while blocking oxygen.

In Example I and Conventional Example I, one of the glass plates was removed, and in Conventional Product (I), both glass plates were used.

Moreover, sample 4 was made of 8 pieces.

After performing electrophoresis for a predetermined time using such a sheet-like electrophoretic membrane, each sheet-like electrophoretic membrane was
The separated electrophoretic images were visualized by staining with ie Blue R.

Experimental Results In Example I, the time required to coat eight samples was approximately 2 minutes, and the resolution of the obtained electrophoretic images was good.

Conventional Example I is a sample held in a sample application jig (approximately 2IJ1
) could not be applied to the sheet electrophoretic membrane (approx. 0.3 m coating). Therefore, the resolution of the electrophoretic image was also extremely low.

In Conventional Example (2), the time required to coat eight samples was approximately 8 minutes, and the resolution of the obtained electrophoretic image was as good as in Example (2).

From the above experimental results, it is clear that in the method of the present invention, the coating operation is very easy and a sufficient electrophoretic image can be obtained.

〔Effect of the invention〕

In the present invention, by applying a voltage to the sheet-shaped electrophoretic membrane while applying the sample, the sample is attracted to the sheet-shaped electrophoretic membrane, so that a sufficient amount of the sample can be reliably applied. , good resolution can be obtained.

Further, the sample application operation is easy and the time required for the application operation can be shortened.

[Brief explanation of the drawing]

Fig. 1 is a perspective view showing the sample application method in electrophoresis of the present invention, Fig. 2 is a cross-sectional view of the same, Fig. 3 is a perspective view of a sample application jig used in the above, and Figs. 4 to 7 are FIG. 8 is a perspective view of another example of a sample application jig, FIG. 8 is a front view of a multiple sample application jig, and FIG. 9 is a perspective view showing a conventional sample application method. 4... Sample 11... Sample application jig 12... Sheet electrophoretic membrane 13... Cathode terminal 14... Anode terminal 18... Electrical connection terminal Figure 3 Figure 4 (N. Figure 8 II II II II l
Figure 9-1

Claims (1)

[Claims] In a method of applying a sample to a sheet-like electrophoretic membrane having a hydrous gel, the sample is held in an electrically conductive sample application jig, and the sample application jig is connected to a cathode, and the sheet-like electrophoresis membrane is connected to an anode. A method for applying a sample in electrophoresis, characterized in that the sample is applied to a sheet-like electrophoresis membrane while applying a voltage between the sample and the sheet-like electrophoresis membrane.