JPH01457A - electrophoresis equipment - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is a method for separating and analyzing polymeric substances, such as proteins and nucleic acids, which can be charged in solution, based on the charge of the molecules and the difference in molecular layers. This invention relates to electrophoresis equipment used for electrophoresis.

[Prior art] An electrophoresis method in which proteins, nucleic acids, their decomposition products, etc. are separated and analyzed based on differences in molecular charge and molecular weight in a membrane-like medium such as a gel membrane or filter paper containing Enokichi solution. is widely known.

In particular, in the field of genetic engineering, techniques for determining the base sequence of radioactively labeled nucleic acids using autoradiography are important. The electrophoretic operation for this purpose is
A base-specific reaction product (mixture) of radiolabeled DNA or DNA fragments is electrophoresed along the direction of an electric field in an electrophoresis medium. At this time, it is common to migrate multiple base-specific reaction products in parallel along the direction of the electric field, and the multiple rows of electrophoretic patterns obtained after electrophoresis are used as autoradiographic images (autoradiograms). After obtaining the results, compare and contrast the migration patterns of each column with each other.
The base sequence is determined.

When using a gel membrane made of starch or polyacrylamide as an electrophoresis medium, conventionally, a flat support such as a glass plate has been used and fabricated by an experimenter each time. This work is extremely troublesome and places a heavy burden on experimenters using electrophoresis. In order to reduce this burden, a spacer with a predetermined thickness is installed at both widthwise ends of two electrically insulating, flexible, and water-impermeable sheets (support sheet and cover sheet). Recently, sheet materials for electrophoresis have become commercially available, in which a gel membrane serving as an electrophoresis medium is housed in a space formed by facing each other.

When performing electrophoresis using such an electrophoresis sheet material, before starting electrophoresis, first remove the upper end of the gel membrane (i.e., the end that does not face the spacer and the upper side of the upright electrophoresis apparatus). It is necessary to inject the sample solution to be electrophoresed into the end (located at the end). Therefore, it is widely practiced to provide a plurality of rectangular wells (also called slots) with one end open near the upper end of the gel film.

However, with this method, one steel plate is placed between adjacent wells.
This spacing creates a gap of the same size between the electrophoresis regions (called lanes) of each sample. As in the case of DNA base sequence analysis, four types of bases (A, G, C,
T) When it is necessary to compare and match the electrophoretic images of each fragment, it is desirable to have no gaps or only a small gap between the electrophoresis lanes, as this gap will make it difficult to compare. .

In order to meet this requirement, a flat member with a serrated protrusion called a shark stay scoum is placed so that it closely or partially penetrates the gel film, and the ends of the gel film and the end surface adjacent to the protrusion are A method is used in which the sample liquid is injected into the approximately triangular space formed by the . In this case, if a comb having the same thickness as the electrophoresis gel membrane is used, the sample liquid often leaks over the partition wall of the comb into the adjacent lane after the sample liquid is injected and before electrophoresis is started.

This phenomenon is particularly likely to occur when a sample solution is injected into a certain lane and the next sample solution is injected after electrophoresis that takes several hours. There are even cases where it becomes impossible.

[Technical Problems to be Solved] The present invention provides for the shark stay scoum to be easily and reliably fixed to the electrophoresis sheet material, and for the sample liquid injection part 11
The purpose of the present invention is to provide an electrophoresis device that does not leak sample liquid from nearby areas.

[Means for Solving the Technical Problems] The above-mentioned problems of the present invention are to provide a gel film serving as an electrophoresis medium to an electrically insulating, flexible, water-impermeable support sheet and a cover sheet, and to their widthwise ends. In an electrophoresis device in which a plurality of sample injection ports are provided in a space formed by a spacer having a predetermined thickness and a plurality of sample injection ports are provided at or near one end of a gel membrane with partition walls interposed between them. A shark-stay scoum having a serrated protrusion is arranged so that the protrusion touches the edge of the gel film, and the approximately triangular space formed by the end of the gel film and the adjacent end surface of the protrusion allows the specimen to be This problem was solved by an electrophoresis device in which the thickness d of the shark's scoum forming the injection port and the partition wall of the sample injection port and the thickness G of the electrophoresis medium gel film have the following relationship (however, The unit is μm).

G×1.1<d<G+50 In the above equation, G is naturally smaller than 500. In sheet materials for electrophoresis, G is usually 140 to 400 (μm
) is within the range.

The thickness d of the shark stay scoum is G×1. l<d≦G+45 It is more preferable if it is within this range.

The thickness d of the comb may be slightly thicker than the thickness of the spacer provided at the ends of the support sheet and cover sheet (usually about 10 to 50 μm thicker than the gel film), but if the difference in thickness is +20 μm It is desirable not to exceed this; if it is thicker than this, it will be difficult to insert the comb.

As a material for the comb, thermoplastic synthetic or semi-synthetic polymers are suitable, but polyethylene terephthalate film is preferred, and although it may be transparent, translucent is preferable for ease of viewing the injection port (for example, manufactured by Lumirror Film@2505IO1$2501410, etc.).

Regarding the length of the comb, if it is short, it will be difficult to handle, and if it is too long, your hand will get caught during operation, so
The width of the comb, which is preferably about 0xz, is of course determined depending on the length of the entire sample entrance to be formed.

The shape of the projections (shark teeth) of the comb can be of any known shape without any particular limitation, but in addition to the shape with a sharp tip, the shape shown in FIG. 1C can be used. Particularly preferred are those having the shape described in Japanese Patent Application No. 190999/1982.

Various known electrophoresis media can be used. As a sheet material for electrophoresis, for example, JP-A-61-
The electrophoretic sheet material using polyacrylamide as the electrophoretic medium described in No. 18852 is suitable. In order to produce such a sheet material for electrophoresis, for example, the method and apparatus described in JP-A-60-203847 can be used.6 The electrophoresis medium used in the present invention may be other than polyacrylamide gel, such as Agarose gel, starch, etc. may also be used.

Polyethylene, polypropylene, polymethyl methacrylate, etc. can also be used as materials for the support sheet.
The same applies to the material of the cover sheet, which is preferably polyethylene terephthalate and whose surface is preferably subjected to hydrophilic treatment using various known methods.

As the electrophoresis device, a known vertical type (upright type) electrophoresis device can be used.
The device described in No. 1 is a preferred example.

A glass plate is usually used to sandwich the electrophoresis sheet material and fix it to the electrophoresis apparatus, but the glass plate may be made of ceramics, quartz, transparent plastic, etc., and can be processed by heat treatment, chemical treatment, etc. The two flat plates are preferably made of tempered glass because they are less likely to break or deform.
They may be made of different materials, but are preferably made of the same material.It is desirable that the two flat plates, such as glass, have a flatness of ±10μ or less.Normally, one of the two flat plates is provided with a buffer. A notch is provided to communicate the liquid bath and the electrophoretic sheet material.

[Effects of the Invention] When the electrophoresis apparatus of the present invention is used, the sample solution does not leak beyond the partition wall of the comb to the injection port of the adjacent lane after the sample solution is injected and before electrophoresis is started. Even if you inject a sample solution into the injection port of a lane and perform electrophoresis, which takes several hours, and then inject the next sample solution into the injection port next to it, such leakage will not occur. will not leak into the adjacent lane between the cover sheet and gel membrane.

Examples are shown below to further specifically explain the present invention.

[Example 1] 1) Acrylamide 7.62N, N-methylenebisacrylamide 0.4y urea in 100111 of gel
The following was added as a polymerization initiator to an aqueous acrylamide solution containing 42 g as well as disodium hydrogen phosphate, sodium dihydrogen phosphate, and tris(hydroxymethyl)aminomethane as buffers.

Beroxonisulfate ammonium 6"; zytetramethylethylenediamine 33μp riboflavin phosphate ester sodium salt 10g2)
The sheet is made of polyethylene terephthalate (PET) with a limited thickness of 175 μm.
) A 250 μl thick film is applied to both ends of a 20 cm wide web (which will serve as a support sheet). Glue a tape-shaped spacer made of polyethylene terephthalate with a width of 10 m,
It was cast into the recesses formed between the spacers to a thickness of 210 μm, and cross-linked and polymerized by irradiation with ultraviolet rays in a nitrogen atmosphere.
A polyacrylamide gel membrane was obtained.

Cut this web into a length of 40 cm, cut only the gel in a straight line 24 mm from one end in the longitudinal direction to remove it, and add a 20 mm deep cut to the exposed end of the PET film.
After making a notch with a width of about 130 mm, a cover sheet made of PET with a shoulder thickness of 63 μm and having the same dimensions as the support was covered to produce an electrophoresis medium sheet.

For comparison purposes, instead of scraping off the gel in a straight line at one longitudinal end of the gel sheet, a strip of 5 mm wide by 5 + a-
An electrophoresis medium sheet was prepared in which a plurality of rectangular notches were provided at intervals of 1.5M steel.

3) An electrophoresis apparatus as shown in No. 111J was assembled using each of the above-mentioned electrophoresis sheets manufactured by Shah Suey Koum.

In order to form a partition wall for a sample injection port at one end of the gel membrane of the electrophoresis medium sheet (the part where only the gel is cut in a straight line), a 250-μl-thick shark scoum (sample injection port) as shown in FIG. Width 4,5 shoulders, bulkhead width 0.2
zz) was inserted to complete the electrophoresis apparatus according to the present invention (no comb was inserted for comparison).

4) After injecting DNA samples labeled with J-F-32p using the Sanger method using microsyringes into the sample injection ports of the two types of electrophoresis instruments mentioned above, and performing electrophoresis on the gel membrane. , autoradiography was performed, and electrophoretic images were obtained for each.

In the electrophoresis image obtained using the electrophoresis apparatus using the shark stay scoum according to the present invention, since adjacent bands are close to each other, it is easy to understand the vertical relationship between them.
The number of bases that could be determined (read) was greater than when using a comparative electrophoresis device.

Furthermore, for comparison, the thickness is 210μl (same thickness as the gel membrane)
When an electrophoresis apparatus was constructed using the Shark Stains scoum and the same operation as above was performed, in many cases the sample solution leaked into the adjacent lane, making it impossible to read the base sequence from the electrophoresis image. Ta.

[Example 2] A polyacrylamide gel membrane was obtained in the same manner as in Example 1, except that a 230-μl-thick spacer was used and the film was molded to a thickness of 200 μl. Add to this a 230 μR thick shark stay scoum (sample injection port width 5 cm, partition wall width 0.5 cm).
zz) was inserted to obtain an electrophoresis device according to the present invention.

As in the case of Example 13'll, an electrophoresis device for comparison having a rectangular groove-shaped sample injection port was prepared.

When electrophoresis was performed in the same manner as in PAt using a DNA sample labeled with ff53 by the Sanger method, the number of bases that could be discriminated (read) in the obtained electrophoretic image was because the numbers of adjacent bands were close to each other. , compared to a comparative electrophoresis instrument that does not use a shark stay scoum and has a Seiaki sample injection port.

For comparison, when we performed the same procedure as above using a shark stew scoum with a thickness of 250 μl (corresponding to the gel film thickness + 50 μl), the sample solution leaked into the gel film around the injection port, and the sample solution was not obtained. The electrophoretic image obtained was unclear.

[Brief explanation of the drawing]

FIG. 1 is an exploded plan view showing an example of the configuration of the electrophoresis device of the present invention, with the support sheet 3 removed. FIG. 1A is a cross-sectional view of the electrophoresis device of the present invention taken along line AA' in FIG. 1, and FIG. 1B is a sectional view taken along line AA' in FIG. 1.
FIG. 1C is a cross-sectional view of the electrophoresis device of the present invention taken along a line, and is a plan view showing an enlarged area within the frame C in FIG. 1. FIG. 2 is a perspective view showing the sample injection section of the electrophoresis apparatus of the present invention into which the shark stay scoum is inserted, and FIG. 3 is an enlarged view showing a situation in which a sample liquid is injected into the sample injection section. The symbols in the figure indicate the following parts. 1: Cover sheet 2: Electrophoresis medium gel membrane 3: Support sheet 4: Shark teeth scoum 5: Sample injection ports 6a, 6b; Spacer x-y end of gel membrane (electrophoresis start line) = for electrophoresis Media Length Applicant: Fuji Photo Film Co., Ltd. Figure 1 Figure 1A Figure 1B

Claims (1)

[Claims] 1) Electrophoresis occurs in a space formed by two electrically insulating, flexible, water-impermeable sheets and a spacer of a predetermined thickness provided at the widthwise ends of the sheets. An electrophoresis device that has a gel membrane as a medium and a plurality of sample injection ports at or near one end of the gel membrane, and has a shark's tooth scoum with a serrated protrusion on the protrusion. is arranged so that it is in contact with the edge of the gel membrane, and the sample injection port is formed by a substantially triangular space formed by the end of the gel membrane and the adjacent end surface of the protrusion, and the thickness d of the shark's teeth scum is An electrophoresis device characterized in that the thickness G of the gel film has the following relationship. G×1.1<d<G+50 (unit: μm) 2) The electrophoresis device according to claim 1), in which the shark's teeth scoum is made of polyethylene terephthalate. 3) The electrophoresis device according to claim 1, wherein the shark's teeth scoum is made of translucent polyethylene terephthalate. 4) The spacer is thicker than the gel film, and the difference between the thickness d of the shark teeth scoum and the thickness of the spacer is +
The electrophoresis device according to claim 1), wherein the electrophoresis device is within 20 μm.