JPS60224050A - 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 constituting the base in such a way that recesses are shown on both sides of the sectional shape of the base in the transverse direction along one surface thereof. CONSTITUTION:The support for the purpose of a supporting medium 21 for electrophoresis is constituted of at least a sheet of a base 20 and is so constituted than the sectional shape of the base 20 in the transverse direction shows, for example, the arc-shaped projecting part having a straight part near the central part. The supporting medium 21 to be formed on the base 20 is made thicker in both side parts along the migration direction thereof than in the central part. The Joule heat enough to compensate the heat radiation from both sides which is regarded to be the major cause for the smiling effect is thus generated on both sides of the medium 21.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to supports for forming and holding electrophoretic support media.

[Background of the Invention] Proteins and protein decomposition products are produced in a sheet-like support medium such as a gel membrane or filter paper impregnated with a buffer solution.

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 or the like is used, and it is in the form of a sheet with a uniform thickness.

These support media consist of a support frame (support) around a flat support (support) made of a glass plate, plastic film, etc.
A gel preparation solution is introduced onto a mold formed by installing a spacer, and if necessary, the upper surface is further sealed with another support, and then the gel is formed.

However, when the above electrophoresis operation is performed using the rectangular sheet-like support medium of uniform thickness formed in this way, even if substances have the same molecular weight, their migration positions differ. Due to the difference in the electrophoresis distance, the phenomenon of uneven migration tends to occur. In other words, the moving speed of charged substances (i.e., the moving distance of 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 as illustrated in Figure 1. , the pattern tends to be such that the moving distance of the zone on both sides is shorter than the moving distance of the zone on the central part. In addition, FIG. 1 schematically shows the electrophoresis pattern of zones 13 and 13' obtained by electrophoresis from the starting point 12 on a support medium 11 formed on a conventional support (support) 10. It shows. This phenomenon is called the smiling effect, and is a major cause of a decrease in the accuracy of the information obtained, such as the base sequence, in operations that include the process of comparing and contrasting multiple electrophoresis columns, such as the determination of DNA base sequences. It becomes.

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.

In the present invention, the support is composed of at least one sheet-like support, and the support is configured such that the cross-sectional shape along the width direction of one surface thereof shows recesses on both sides. The present invention provides a support for a support medium for electrophoresis, characterized in that the support medium includes:

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 present invention will be described below with reference to the accompanying drawings.

A support for an electrophoresis support medium provided by the present invention includes at least one support, and recesses are provided on both sides of the support along the electrophoresis direction on the side that contacts the support medium. Therefore, the support medium for electrophoresis formed on the support using this support can have a structure in which the thickness of both sides along the migration direction is thicker than the thickness of the center part. . When electrophoresis is performed using a support medium with such a configuration, a sufficient amount of Joule heat is released from both sides of the support medium to compensate for the heat dissipation from both sides, which is considered to be the main cause of the smiling effect. As a result, the migration speed of the electrophoretic substance on both sides increases, thereby preventing or reducing the smiling effect.

The support constituting the support for the support medium for electrophoresis of the present invention can have a shape as shown in FIG. 2, for example. That is, the shape of the support body 20 in the length direction and the width direction is similar to the conventionally used support body (10) as shown in FIG. 1, but the cross-sectional shape in the width direction is It is configured to exhibit an arcuate convex portion with a straight portion near the center. Therefore, the support medium 21 formed on this support 20 has the same shape in the length direction and width direction as the conventional support medium (11) in FIG. ) is thicker on both sides than in the center.

Electrophoresis operation is the 25th! This is carried out by respectively immersing the upper and lower ends of the support medium 21 held on the support 20 of J in a buffer solution that is in contact with the electrode according to a conventional method. A charged substance whose migration has started from the migration start point 22 shows a migration pattern after a certain period of time, but the migration pattern developed on the support medium 21 is in the zone 23.
．． 23' indicates a straight line or a pattern similar to a straight line. 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 may have a cross-sectional shape having a concave portion with a straight bottom on both sides, for example, it may take the cross-sectional shape shown in FIGS. 3a and 3b. possible (allbl: support, a2 + b2
: supporting medium).

As another aspect, when the width of the support is short, its cross-sectional shape may exhibit a convex portion, and the surface that contacts the support medium may be a convex surface, for example, the 30.3d
The cross-sectional shape shown in the figure can be taken (Ci, ds:
support, 02 + d2: support medium). That is, the change in thickness (change in width) of the support may be continuous or stepwise.

In the present invention, the surface of the support that comes in contact with the support medium may have the above shape, and the other portions, such as the back surface, do not necessarily need to be flat.

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 above-described cross-sectional shape.

Alternatively, as shown in FIG. 3e, both cross sections of the two supports may have the above shapes (eI, e3:
support, e2: 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 support It is sufficient if the cross-sectional shape is as described above.

Note that the support constituting the support of the present invention does not need to have a completely flat surface in contact with the support medium in its length direction; for example, as shown in FIG. and both ends) may have a shape in which four parts are provided with a constant width.

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. For example, a support made of a support with a convex surface, which is one embodiment of the present invention, can be obtained by deforming a plastic film by heat processing or the like. Alternatively, the support material may be introduced into a suitable mold and molded. Further, a desired support may be obtained by subjecting a molded product such as a sheet-like material to processing such as cutting or grinding. Furthermore, a spacer having an appropriate thickness and shape may be attached to a sheet-like object to be integrated.

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, another support may be placed on the mold to seal it, or a slot former may be placed on top of the gel to provide a number of slots for sample introduction. You may.

In this way, a gel support medium is obtained in which the thickness on both sides along the length is greater than the thickness in the center.

When manufacturing the support of the present invention, the preferable step difference between the concave portions on both sides and the central portion of the support or the preferable curvature of the convex surface 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, considering the degree of the smiling effect from the electrophoresis pattern developed on the support medium, that is, the difference between the migration distance in the center and the migration distance on both sides, a recess (on both sides) and/or a convex surface is formed on the support. (other than both measuring sections) will be provided. 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.

Note that during electrophoresis operation, the support of the present invention does not necessarily need to be held in close contact with the support medium, and only the support medium may be installed in the electrophoresis apparatus after separating the support medium from the support.

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

[Comparative Example 1] Two colorless and transparent polyethylene terephthalate sheets (
Glow discharge treatment (200cm, 1.OA) was applied to the surfaces of each substrate (4'Ocm long, 20cm wide, 180pm thick) to obtain two supports whose surfaces were made hydrophilic.

A polyethylene terephthalate tape (with adhesive layers on both sides, width 10 mm, total thickness including the adhesive layer 0.5 mm) was placed around the single support whose surface had been 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), and agarose (gelling temperature 36°C,
Low electroosmotic type) 0.3g, polyacrylamide 2
．． 5g, disodium hydrogen phosphate φ dodecahydrate 3.58g
, sodium dihydrogen phosphate dihydrate 0.33 g, and SDS (sodium dodecyl sulfate) 0. 1.3 mJl of ammonium belloxonisulfate (5*wt% aqueous solution) as a polymerization initiator to 100ml of an aqueous solution consisting of 10g
A gel preparation solution containing 33 PM of TEMED (N, N, N', N'-tetramethylethylenediamine) was cast and gelated to form a polyacrylamide gel film with a thickness of 0.5 mm. 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.

Using the support medium described above, 1.05 g of tris(hydroxymethyl)aminomethane, 0.55 g of boric acid and EDT.
Using an aqueous solution made by adding water to 93 mg of A-2Na to make joomJl as a buffer solution, bromophenol blue (CAS Registry N
o, [115-39-91) was electrophoresed 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] A colorless and transparent polyethylene terephthalate sheet (length 40 cm, width 20 cm, thickness 1807 tm) whose surface was subjected to hydrophilic treatment in the same manner as in Comparative Example 1 was placed in the center of the surface. A support as shown in FIG. 4 was prepared by adhering a colorless and transparent polyethylene terephthalate spacer sheet (27 cm long, 14 cm wide, 180 pm thick) to form a trapezoidal convex portion in the center of the sheet. In addition, the same support as in Comparative Example 1 was also prepared.

Place the support with the surface with the trapezoid facing upwards, and tape polyethylene terephthalate tape (with an adhesive layer on both sides, width 10 mm, total thickness including the adhesive layer) on the support. 0.5 mm) was attached as a spacer to form a mold.

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.

Electrophoresis of bromophenol blue was carried out for t-3 hours under the same conditions as in Comparative Example 1 using the support medium of ]-2.

The migration pattern after electrophoresis showed a nearly straight line as shown in FIG. 2, and the occurrence 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. 10.20: Support (support) 11.21: Support medium for electrophoresis 12.22: Starting point of electrophoresis 13.13', 23.23'': Migration pattern (zone) 3a, 3b, 3c, 3d and 3c are diagrams showing other examples of cross-sectional shapes in the width direction of the support of the present invention. al, bl, C1, dl, el, C3: supports a2, b
2, C2, dl, C2: Support medium FIG. 4 is a diagram showing another example of the support of the present invention. Figure 3q

Claims (1)

[Claims] l. The support is composed of at least one sheet-like support, and the support is configured such that one surface of the support has a cross-sectional shape along the width direction showing recesses on both sides. A support for a support medium for electrophoresis. 2. Electrophoresis according to claim 1, wherein the cross-sectional shape in the width direction of the 2° support is configured to exhibit an arcuate convex portion having a straight portion near the center. Support for supporting media. Claim 1 or 2, characterized in that the 3° support is composed of two sheet-like supports, and at least one of the supports has a cross-sectional shape. Support for the described electrophoretic support medium. Claim 1, wherein the 4° support is composed of two sheet-like supports and a support frame, and at least one of the supports has the above-mentioned cross-sectional shape.
A support for the electrophoresis support medium according to item 1 or 2. 5. A support for an electrophoretic support medium according to claim 1, characterized in that the 5° support is made of glass or plastic material. 6. A support for a support medium for electrophoresis according to claim 1, wherein the support medium is a gel membrane.