JPS60224049A - Support for supporting medium for electrophoresis - Google Patents

Abstract

PURPOSE:To prevent or decrease a smiling effect by constituting a support of at least a sheet of sheet-shaped base and making the length on both sides thereof along the migration direction shorter than the length in the central part. CONSTITUTION:The support for a supporting medium for electrophoresis is constituted of at least a sheet of a base 21 and the length on both sides of the base 21 along the migration direction is made shorter than the length in the central part by forming, for example, one end into an arc shape. The electrically charged material which starts migration from a start point 22 for migration on the base 21 exhibits the migration pattern of zones 23, 23' after specified time and the potential gradient on both sides of the base 21 is larger than that in the central part in this stage and therefore the moving speed of the charge material near both ends is relatively increased and the decrease in the moving speed owing to Joule heat radiation is prevented.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a support for forming or holding an electrophoretic support medium.

[Background of the Invention] Proteins and protein decomposition products are produced in sheet-like support media such as gel membranes and synthetic papers impregnated with buffer solutions.

Electrophoresis operations have been known for some time, and their main purpose is to separate charged molecules or particles, such as nucleic acids and nucleic acid decomposition products, by utilizing the phenomenon in which they move under the influence of an electric field. It is used for the separation and identification of biopolymer substances such as.

Especially in the field of genetic engineering, which has recently attracted attention,
In order to determine the base sequence of a nucleic acid such as DNA using autoradiography, it is essential to perform an electrophoretic operation. Electrophoresis operations for this purpose generally include operations in which multiple types of radioactively labeled DNA or base-specific cleavage mixtures of DNA fragments are run in parallel along the length of an electrophoresis support medium. It will be done. Then, a multi-row migration pattern (
After obtaining an autoradiograph (a collection of zones formed on a support medium by electrophoresis), the base sequence is determined by comparing the zones in each row with each other. That is, the comparison is made using the principle of electrophoresis that base-specific cleavage products having the same molecular weight move to the same position if the starting point of electrophoresis is the same.

Generally, starch gel membranes are used as support media for electrophoresis.
A polyacrylamide gel membrane is used, and it is shaped like a rectangular sheet. These support media are prepared by introducing the gel preparation onto a mold formed by installing a support frame (spacer) around a rectangular support (support) made of a glass plate, plastic film, etc. It is formed by sealing the top surface with another support and then gelling it.

However, when the above-mentioned electrophoresis operation is performed using the rectangular sheet-like support medium formed in this way, even substances having the same molecular weight are separated due to differences in their migration positions. The phenomenon of uneven migration distance is likely to occur. In other words, the moving speed of charged substances (
In other words, the migration distance of the zone (in other words, the distance traveled by the zone) generally tends to be smaller on both sides than in the center, and therefore, the migration pattern after a certain period of time is the migration distance of the zone on both sides, as illustrated in Figure 1. However, the pattern tends to be shorter than the moving distance of the zone in the center. In addition, FIG. 1 schematically shows the migration pattern of zone 13.13' obtained by electrophoresis from the starting point 12 on the support medium 11 formed using a conventional support (support). It is something. This phenomenon is called the smiling effect, and D
In an operation that includes a step of comparing and contrasting multiple electrophoresis columns, such as an operation for determining a base sequence such as NA, this is a major cause of a decrease in the accuracy of the information obtained such as the base sequence.

The main reason for the above-mentioned smiling effect is that the heat (Joule heat) generated by current passing through the support medium is dissipated from the side edges, resulting in a temperature difference between the center and side surfaces. There are many things that can happen. That is, the generated Joule heat is radiated only from the upper and lower surfaces in the center of the support medium, but in the side portions, heat is further radiated from the side edges. Note that movement of Joule heat along the width direction occurs to compensate for these heat radiations, but even with regard to this heat movement, the heat radiation is likely to be compensated for in the central part of the support medium by the inflow of heat from both sides. In both side portions, heat only flows in from one side, resulting in insufficient compensation for heat radiation. For these reasons, the temperature at the side portions tends to be lower than that at the center, and as a result, the moving speed of charged substances at the lower temperature side portions is lower than that at the center, causing them to appear on the migration pattern. produces a smiling effect.

Conventionally, in order to prevent the smiling effect from occurring, measures such as attaching a heat sink to the entire surface of the support medium to suppress the generation of temperature differences have been taken, but such measures are insufficient to achieve the smiling effect. It is difficult to prevent this, and the work of installing a heat sink is also complicated.

[Summary of the Invention] The present invention provides a support for a support medium for electrophoresis that makes it possible to prevent or reduce the smiling effect that often occurs during electrophoresis operations in which a large number of electrophoresis rows are formed on a single support medium. Its purpose is to provide.

The present invention provides that the support is composed of at least one sheet-like support, and that the length of both sides along the migration direction of the support is shorter than the length of the center. The present invention provides a support for a supporting medium for electrophoresis.

Note that in the present invention, the term "supporter" basically means a supporter for holding a support medium, but the present invention also includes a supporter including a support frame used for forming a mold (mold forming kit). Included in the support. In addition, in the present invention, both sides of the support constituting the support means portions corresponding to both sides of the effective zone of the support medium, that is, the entire zone in which a plurality of migration rows are formed, and does not necessarily mean It does not necessarily mean both sides of the support itself.

[Detailed description of the invention] The invention will now be described with reference to the accompanying drawings.

A support for an electrophoresis support medium provided by the present invention includes at least one support, and the length of both sides of the support along the migration direction is shorter than the length of the center. Therefore, the support medium for electrophoresis formed on the support using this support can be configured such that the length of both sides along the migration direction is shorter than the length of the center part. .

When performing electrophoresis by applying a constant voltage to both ends of a support medium with such a configuration, the length of both sides is shorter than the length of the center, so the potential gradient (in the direction of migration) on both sides increases. (potential gradient along the center) can be made larger than the potential gradient at the center. The speed of movement of a charged substance during electrophoresis is basically directly proportional to the potential gradient of the electric field near where the charged substance is located, so the speed of movement of a charged substance moving close to both sides and the center are The difference between the moving speeds of identically charged substances becomes zero or is at least reduced, and the smiling effect is prevented or reduced.

The support constituting the support for the support medium for electrophoresis of the present invention has, for example, an arcuate shape at one end in the length direction with a straight portion near the center. (See Figure 5). Therefore, as shown in FIG. 2, the support medium formed using this support tool has an arcuate shape in which the lengthwise (migration direction) ends of the support medium 21 have a straight portion near the center. have.

In the electrophoresis operation, as shown in FIG. 3, another support 34 of the same shape is stacked and tightly sandwiched on the opposite side of the support medium 31 to the side in contact with the support 34, and then the support medium is This is carried out by immersing the upper end and the lower end in a buffer solution in contact with the electrode in a conventional manner. As shown in FIG. 2, the charged substance whose migration starts from the migration start point 22 on the support medium 21 shows a migration pattern after a certain period of time, but the migration pattern developed on the support medium 21 is .23' indicates a straight line or a straight-line-like turn.

A constant voltage is applied in the length direction of the support medium by the above pair of electrodes, but the electrode spacing on both sides of the support medium (effective electrode spacing □, that is, the surface of each buffer in contact with the support medium) Since the distance between the electrodes (distance between the electrodes) is smaller than the electrode spacing at the center, the potential gradient on both sides of the support medium is larger than the potential gradient at the center, and as a result, the moving speed of the charged substance near the two measuring sections is relatively It is possible to prevent the movement speed from decreasing due to the radiation of Joule heat at the side edges. That is, when electrophoresis is performed using a support medium formed by the support of the present invention, the occurrence of the smiling effect, which has been a problem in the past, is prevented or at least reduced.

The support constituting the support of the present invention only needs to have the lengths of both sides along the electrophoresis direction shorter than the length of the center. For example, as shown in FIGS. 4a and 4b, the lower end thereof is It is also possible to have a shape in which the support body has a straight portion near the center, and the lengths of both sides of the support body are shorter than the length of the center portion.

Further, when the width of the support is short, the lower end thereof can be formed into an arc shape as shown in FIG. 4c. That is, the change in length (change in width) of the support may be continuous or stepwise.

Further, the shape shown in "2" in which the length in the migration direction is changed may be formed not only at the lower end of the support but also at the upper end. Note that when only the shape of one end of the support has changed, electrophoresis does not necessarily have to start from the flat side of the end.

The support constituting the support of the present invention may be further configured such that its cross-sectional shape in the width direction shows recesses on both sides along the electrophoresis direction, as shown in FIG. 5a or 5b. . A support medium formed using a support having such a shape not only has the length of both sides along the electrophoresis direction shorter than the length of the center, but also has a thickness of both sides that is shorter than the center. Since it is thicker than the thickness of , it is possible to effectively prevent a temperature drop due to the radiation of Joule heat, and therefore, it is more effective in preventing or reducing the smiling effect.

In another embodiment, the support of the present invention may be composed of two supports. In that case, at least one of the supports may have the shape described above. However, when forming a support medium using the support of the present invention, it is necessary to use a support having the above shape for mold formation.

Then, by forming a support medium on this support, a support medium having the above shape can be obtained.

The electrophoresis operation is usually carried out with a support layered on the other side of the support medium and closely sandwiched between them, as shown in Figure 3. The other support may be a conventional rectangular shape (however, it is necessary to cover the edges of the support medium).

Furthermore, the support of the present invention may be a kit for forming a mold of a support medium for electrophoresis, which includes two supports and a support frame (spacer) provided between them, in which case at least one of the supports is It suffices if it has the shape described above.

As the material used for the support, conventionally known materials can be used, such as glass; plastic materials such as acrylic resin and polyethylene resin. The support of the present invention can be manufactured by molding these materials by a known method.

Examples of support media for electrophoresis formed using the support of the present invention include cellulose acetate membranes, starch gel membranes, and polyacrylamide gel membranes. Although the support of the present invention can be used to form any electrophoretic support medium, polyacrylamide gel membranes are particularly preferred.

A support medium for electrophoresis can be formed using the support of the present invention, for example, as follows.

A support frame (
On the mold formed by installing a tape-shaped spacer),
It is formed by introducing a gel preparation solution made of acrylamide or the like and then gelling it. During gelation, after introducing the gel preparation solution, a single support may be placed on the mold to seal it, or a slot former may be placed on top of the gel to form multiple slots for sample introduction. may be provided.

In this way, a gel support medium is obtained in which the length of both sides along the migration direction is shorter than the length of the central part.

In manufacturing the support of the present invention, a preferable difference between the lengths of both side parts and the length of the central part of the support or a preferable curvature can be determined experimentally. For example, first, a rectangular support medium of uniform thickness is formed using a conventional support made of a sheet-like support, and electrophoresis is performed using this under predetermined conditions. Then, the extent of the smiling effect, that is, the difference between the migration distance in the center and the migration distance on both sides, is measured from the migration pattern developed on the support medium, and this difference or a value close to this difference is measured on both sides of the support. The preferred difference between the length of the section and the length of the central section is that the support is shaped to match that difference. A support medium is formed using this support in the same manner as described above, and electrophoresis is performed again using this under the same conditions. By repeating such experiments, it is possible to understand the correlation among the gel composition, dimensions of the support medium, electrophoresis conditions, etc., and based on this, it is possible to create a support with a suitable shape.

In addition, the support of the present invention does not necessarily need to be held in close contact with the support medium during the electrophoresis operation, and after separating the support medium from the support, the electrophoresis operation may be performed while being sandwiched between, for example, another support. Good too.

Further, the support of the present invention can also be used only for closely holding a support medium that has already been formed during electrophoresis. For example, as shown in FIG.
The electrophoresis operation may be performed in a state where the samples are tightly sandwiched at 4.64°. Since the end surface of the support that comes into contact with the buffer during electrophoresis becomes an equipotential surface, the smiling effect can be prevented or reduced in the same way as when the shape of the support medium itself is changed.

In this case, the support of the present invention can be applied to conventionally known support media for electrophoresis, such as filter paper, in addition to the support media exemplified above.

Next, the present invention will be explained with reference to Examples and Comparative Examples.

[Comparative Example 1 Two colorless and transparent polyethylene terephthalate sheets (
Glow discharge treatment (200V, 1.OA) was applied to each surface of each support (length: jocm, width: 20cm, thickness: 180l) to obtain two supports whose surfaces were made hydrophilic.

Polyethylene terephthalate tape (with adhesive layers on both sides, width 10 mm, total thickness including adhesive layer 0.5 mm) was placed on both sides of the single support whose surface was made hydrophilic as described above. ) was attached as a spacer to form a mold. Next, 9.5 g of acrylamide, 0.5 g of BIS (N,N''-methylenebisacrylamide), 0.3 g of agarose (gelling temperature 36°C, low electroosmotic type), and 2.5 g of polyacrylamide were placed in this mold.
5g, disodium hydrogen phosphate - 3.58g of water salt,
Sodium dihydrogen phosphate, industrial water salt 0.33g, and S
To 100 mJL of an aqueous solution consisting of 0.10 g of DS (sodium dodecyl sulfate), 1.3 mJL of ammonium belloxonisulfate (5% by weight aqueous solution) as a polymerization initiator,
A polyacrylamide gel film having a thickness of 0.5 mm was formed by casting and gelling a gelling solution to which 33% of TEMED (N, N, N', N'-tetramethylethylenediamine) was added. Next, 17 sample injection slots were formed at equal intervals on one end of this gel membrane, and another support was placed on top of the gel membrane, attached with spacers, and electrically applied. A support medium for migration was obtained.

Tris(hydroxymethyl)
1.05g aminomethane, 0.55g boric acid and ED
Bromophenol blue (CAS Registry No.
Electrophoresis of [115-39-9) was performed for 3 hours.

A smiling effect as shown in FIG. 1 appeared in the migration pattern after electrophoresis, and there was a difference of about 3 cm between the migration distance in the center and the migration distance on both sides.

[Example 1] Two colorless and transparent polyethylene terephthalate sheets (length 40 cm
, width 20 cm, thickness 180 lm) was prepared.

Polyethylene terephthalate cheese (with adhesive layer on both sides, width 10 mm, total thickness including adhesive layer 0', 5 mm) is placed on both sides of the sheet whose surface has been made hydrophilic as described above. ) and used it as a spacer. Next, one end of this sheet (the end opposite to the side that becomes the starting point of electrophoresis) was cut into an arc shape with a straight section of 8 cm near the center, and the length of the center and both sides were cut. Supports having different lengths (however, the difference between the length of the center part and the length of both sides was 4 cm) were obtained, and a mold was formed.

Next, a gel preparation solution was cast into this mold in the same manner as in Comparative Example 1, and gelatinized to form a polyacrylamide gel membrane. After forming 17 sample injection slots at equal intervals on one end of this gel membrane, another support was placed on top of the gel membrane, attached with spacers, and electrically connected. A support medium for migration was obtained.

Bromophenol blue electrophoresis was performed for 3 hours under the same conditions as in Comparative Example 1 using the above support medium.

The migration pattern after electrophoresis showed a nearly straight line as shown in FIG. 2, and the origin of the smiling effect was negligible.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the smiling effect of a migration pattern developed on a support medium for electrophoresis formed using a conventional support. FIG. 2 is a schematic diagram showing a migration pattern developed on an electrophoresis support medium formed using the support of the present invention. FIG. 3 is a partial perspective view showing an example of the structure of the support device and support medium of the present invention in electrophoresis operation. 4a, 4b, and 40 are diagrams showing examples of other shapes of the support of the present invention. Figures 5a and 5b are views showing another example of the support of the present invention. FIG. 6 is a partial perspective view showing an example of the structure of the support device and support medium of the present invention in electrophoresis operation. 11.21.31.61: Support medium for electrophoresis 12.2
2: Starting point of electrophoresis 13.13', 23.23": Electrophoresis pattern (zone) 34.34", 64.64': Support (support) Figure 1 Figure 2

Claims (1)

[Claims] l. An electrical device characterized in that the support is composed of at least one sheet-like support, and the length of both sides along the migration direction of the support is shorter than the length of the center part. Support for migration support medium. 2. A support for an electrophoretic support medium according to claim 1, wherein at least one end of the 2° support has an arcuate shape with a straight portion near the center. . Electrophoresis according to claim 2, characterized in that the end of the 3° support opposite to the electrophoresis starting point side has an arcuate shape with a straight portion near the center. Support for supporting media. Any one of claims 1 to 3, wherein the 4° support is composed of two sheet-like supports, and at least one of the supports has the above shape. A support for the electrophoresis support medium described in the section. 5. Claims 1 to 3, characterized in that the support is composed of two sheet-like supports and a support frame, and at least one of the supports has the above shape. A support for the support medium for electrophoresis according to any one of the items. 6. A support for an electrophoretic support medium according to claim 1, characterized in that the support is made of glass or plastic material. 7. A support for a support medium for electrophoresis according to claim 1, wherein the support medium is a gel membrane.