JP4362659B2 - Electrophoresis apparatus and electrophoresis method - Google Patents

Description

  The present invention relates to an electrophoresis apparatus and method, and more particularly to an electrophoresis apparatus and method for separating a substance to be separated in a medium.

  In the field of life science, electrophoresis technology is one of very useful separation and analysis techniques. In addition to being used for separation of proteins, DNA, RNA and the like that are biopolymers, it is also possible to separate cells and the like. So far, in addition to typical electrophoresis using gels, capillary electrophoresis using a buffer solution containing a polymer, free flow electrophoresis in which electrophoresis is performed in a free solution, and the like have been performed.

Patent Document 1 describes, as a technique for improving the electrophoresis technique, a wire-like conductive material is provided in the electrophoresis tank to eliminate disturbance of the electrophoresis pattern due to unevenness of the electrophoresis electric field. Patent Document 2 describes that an intermediate electrode is provided in the migration tank to actively control the electric field and potential distribution inside the migration tank.
Japanese Utility Model Publication No. 5-4004 (released on January 22, 1993) Japanese Utility Model Publication No. 5-4003 (released on January 22, 1993)

  However, as a result of investigations by the present inventors, the electrophoresis speed is low in the electrophoresis method using the electrophoresis apparatus having a conductor in the electrophoresis tank as described in Patent Documents 1 and 2. I found that there is a problem.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to suppress a decrease in migration speed in an electrophoresis apparatus provided with a conductor in the migration tank.

  As a result of intensive studies, the inventors of the present invention have found that the cause of the decrease in the migration speed is that bubbles generated in the conductor in the electrophoresis tank peel off the gel from the conductor, and the gap between the gel and the conductor. It has been found that this is caused by a phenomenon that has not been known at all, that the buffering action is inhibited by the penetration of the buffer solution.

  The inventors have further studied and found that bubbles do not occur if the potential difference between adjacent conductors in the electrophoresis tank is not more than a certain value, thus completing the invention.

  That is, the electrophoresis apparatus according to the present invention is an electrophoresis apparatus that separates a substance to be separated in a medium disposed in contact with a plurality of conductors, and includes a voltage applying unit that applies a voltage to the medium. The voltage applying means applies the voltage so that the potential difference between the adjacent conductors is greater than 0V and less than or equal to 0.3V.

  According to said structure, a voltage application means applies a voltage so that the electrical potential difference between the said adjacent conductors may become larger than 0V and below 0.3V. If the potential difference between the adjacent conductors is 0.3 V or less, no bubbles are generated as shown in the examples described later. Therefore, according to said structure, the fall of the migration speed resulting from bubble generation can be suppressed.

  In the electrophoretic device, the voltage applying unit may apply the voltage to the conductors.

  According to the above configuration, since the voltage applying unit directly applies a potential to each of the conductors, the potential difference between the adjacent conductors can be easily set to be greater than 0V and less than or equal to 0.3V. can do.

  In the electrophoresis apparatus, the number of the plurality of conductors is preferably 250 or more.

  According to said structure, even if the applied voltage (50-500V) with respect to the gel of the range normally used for electrophoresis, the electrical potential difference between adjacent conductors can be made easy to 0.3V or less.

  In the electrophoretic device, it is preferable that the conductor is a linear conductor.

  According to said structure, since a linear conductor with a narrow width | variety is used, many conductors can be arranged and the electrical potential difference between adjacent conductors can be made easy to 0.3V or less.

  In the electrophoretic device, it is preferable that the linear conductors are arranged in parallel with each other so that the voltage applying unit is orthogonal to the direction in which the voltage is applied to the medium.

  According to the above configuration, the linear conductors are arranged in parallel with each other so that the voltage applying unit goes straight in the direction in which the voltage is applied to the medium. Therefore, the conductor hardly affects the potential gradient in the first direction. Therefore, the substance to be separated contained in the medium can be suitably separated.

  In the electrophoresis apparatus, it is preferable that a width of the linear conductor is 100 μm or less.

  According to the above configuration, since the width of the linear conductor is narrow, a large number of conductors can be arranged even in a small-scale device, and the potential difference between adjacent conductors can be reduced to 0.3 V or less. Can be easily.

  In the electrophoretic device, the distance between the linear conductors is preferably 100 μm or less.

  According to said structure, since the space | interval of the said linear conductors is narrow, even if it is a small apparatus, many conductors can be arrange | positioned and the electric potential difference between adjacent conductors is 0.3 V or less Can be easily.

  In the electrophoresis apparatus, the conductor is preferably made of a metal selected from the group consisting of platinum, zinc, and copper.

  According to said structure, since the said conductor is hard to deteriorate, the electrophoresis method which concerns on this invention can be performed repeatedly.

  The electrophoretic device may be an isoelectric focusing device.

  According to the above configuration, in the isoelectric focusing device, a uniform pH gradient can be formed in the medium.

  An electrophoresis method according to the present invention is an electrophoresis method for separating a substance to be separated in a medium placed in contact with a plurality of conductors, wherein a potential difference between adjacent conductors is greater than 0V and It includes a step of applying a voltage to the medium so that the voltage is 0.3 V or less.

  According to said structure, in a voltage application process, a voltage is applied so that the electrical potential difference between the said adjacent conductors may become larger than 0V and below 0.3V. If the potential difference between the adjacent conductors is 0.3 V or less, no bubbles are generated as shown in the examples described later. Therefore, according to said structure, the fall of the migration speed resulting from bubble generation can be suppressed.

  ADVANTAGE OF THE INVENTION According to this invention, generation | occurrence | production of the bubble in the electrophoresis in the conductor in an electrophoresis tank can be suppressed, and the fall of an electrophoresis speed can be suppressed.

  An embodiment of an electrophoresis apparatus according to the present invention will be described with reference to FIG.

  The electrophoresis apparatus 100 according to this embodiment includes buffer solution tanks 101 and 102, a separation unit 103, electrodes 104 and 105 (voltage applying means), a stripe electrode 106 (conductor), and a lid member 107. Electrodes 104 and 105 are disposed in the buffer baths 101 and 102, respectively, and are filled with a buffer solution. The separation unit 103 exists between the buffer solution tanks 101 and 102. A stripe electrode 106 is provided on the separation unit 103, and the medium 120 is placed on the stripe electrode 106 of the separation unit 103.

  As the buffer solution, a buffer solution having a composition generally used for electrophoresis may be used. The medium 120 is a gel generally used for electrophoresis, such as an agarose gel or a polyacrylamide gel, and contains a substance to be separated. The substance to be separated may be a substance to be separated or analyzed by electrophoresis and transcription, and a preparation from a biological material (for example, an individual organism, body fluid, cell line, tissue culture, or tissue fragment) is preferably used. be able to.

  The electrode 104 and the electrode 105 apply a voltage to the medium 120 so that the potential difference between the conductors of the stripe conductor 106 is 0.3 V or less. Thereby, generation | occurrence | production of a bubble can be prevented. Specifically, the voltage applied by the electrode 104 and the electrode 105 is determined from the number of conductors included in the stripe electrode 106, and follows the following formula (1). Note that a voltage exceeding 0 V must be applied.

Applied voltage (V) ≦ (Number of conductors −1) × 0.3 (V) (1)
In one aspect, the electrophoresis apparatus 100 according to the present embodiment includes potential control means (voltage application means) (not shown) that controls the potential of each conductor of the stripe conductor 113. The potential control means includes, for example, a power source and a conductive path, and is electrically conductively connected from the power source to each of the conductors via paths having different resistance values or sub power sources. Different potentials are applied to the conductor. Note that it is not always necessary to control the potentials of all the conductors, and only the potentials of some conductors (hereinafter referred to as intermediate electrodes) may be controlled. In this case, the other conductors sandwiched between the intermediate electrodes have a potential according to a potential gradient formed by the intermediate electrodes. In either case, it is only necessary to apply a potential so that the potential difference between the adjacent conductors is 0.3 V or less, and this can prevent generation of bubbles.

  If the potential control means is used, electrophoresis can be suitably performed in addition to the above effects. For example, among the substances to be separated in the medium 120, the low-molecular-weight substance that moves quickly and has a large potential gradient in the low-potential region so as to increase the separation width of the high-molecular-weight substance that is difficult to separate from the medium 120. By separating the potential gradient in the high potential region so as not to leave, a substance to be separated having a large range in the molecular weight of the contained substance can be suitably separated. That is, in electrophoresis, substances migrate from the low potential side to the high potential side, but high molecular weight substances remain on the low potential side, and low molecular weight substances tend to move to the high potential side. Here, as described above, if the potential gradient in the low potential region is increased and the potential gradient in the high potential region is decreased, the degree of separation of the high molecular weight substance is increased and the movement of the low molecular weight substance is increased. Can be suppressed.

  In another embodiment, the electrophoresis apparatus according to the present invention is an isoelectric focusing apparatus. FIG. 2 is a perspective view of the isoelectric focusing apparatus 200 according to the present embodiment, FIG. 3 is a top view thereof, and FIG. 4 is a side view thereof. The isoelectric focusing device 200 includes a separation tank 201, electrodes 202 and 203 (voltage applying means), stripe electrodes 204 and 205, and a lid member 206. The medium 220 is held between the stripe electrodes 204 and 205 in the separation tank 201.

  The medium 220 preferably contains an ampholite for forming a pH gradient. When the electrode 202 and the electrode 203 apply a voltage to the medium 220, the individual conductors in the stripe electrodes 204 and 205 each have one potential and the conductors of the stripe electrode 204 and the stripes having the same distance from the electrodes The conductor of the electrode 205 has the same potential. Therefore, the potential is uniform in the vicinity of the individual conductors of both stripe electrodes, and the amphorite in the medium 220 moves accordingly, and the pH gradient becomes uniform.

  At this time, the electrode 202 and the electrode 203 apply a voltage so that the potential difference between the conductors of the stripe conductors 213 and 214 is 0.3 V or less, so that the generation of bubbles can be prevented.

  In still another embodiment, the electrophoresis apparatus 300 according to the present invention applies a voltage to the medium 320 using the stripe electrodes 301 and 302. FIG. 5 is an explanatory diagram of the operation of the electrophoresis apparatus 300 according to the present embodiment. As shown in FIG. 5, in the electrophoresis apparatus 300, electrophoresis is performed by applying a voltage only between the corresponding adjacent conductors of the stripe electrodes 301 and 302 and sequentially shifting the conductors to which the voltage is applied.

  In addition to applying a voltage to each adjacent conductor, the voltage is applied to the conductors, such as when the conductor pitch is fine. A voltage may be applied and sequentially shifted. For example, in the case of applying 10 V, by setting an interval of 34 or more, it is possible to apply so that the potential difference between the adjacent conductors is substantially 0.3 V or less.

  At this time, generation of bubbles can be prevented by setting the potential difference between adjacent conductors to 0.3V. In the present embodiment, since a potential is applied only to a limited number of electrodes, there are fewer problems caused by the generation of bubbles compared to the other embodiments. However, it goes without saying that the voltage is preferably set to 0.3 V or less.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

[Example 1: Production of stripe electrode]
A Cr binder film was formed on a 6 cm × 5 cm glass plate having a thickness of 3 mm using a sputtering apparatus, and then about 2000 mm of Pt was formed. Next, using a dicing saw (Disco) in which a blade having a width of 100 μm was set, the Pt electrode width and the distance between the electrodes were about 100 μm and about 100 μm. In addition, a strip electrode substrate having a Pt line width of 100 μm and a space between Pt lines of 100 μm and a Pt line width of 50 μm and a Pt line using a CO ₂ laser engraving machine under a cutting condition of 7% laser power, 6% processing speed, and 3000 Hz. A stripe electrode substrate with a gap of 50 μm could be produced (FIG. 6). In addition, the glass surface was hardly subjected to laser processing under these conditions.

[Example 2: Comparative study of substrates]
A 6 cm × 5 cm gel plate (glass plate) was assembled with a 1 mm spacer in between, and placed in a gel preparation container. When a glass substrate is used as one gel plate and a stripe electrode substrate having a Pt line width of 100 μm and a Pt line width of 100 μm as the other gel plate, a substrate on which the entire substrate is formed of Pt, or A glass substrate (positive control) was used.

  Next, the prepared separation gel solution (13% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 378 mM Tris-HCl (pH 8.8), 0.05% APS, 0.1% TEMED) Was added up to 7 mm from the tip, and then water was overlaid. After the separated gel solution was polymerized, water was removed, and a concentrated gel solution (4% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 125 mM Tris-HCl (pH 8.8), 0.8% was prepared. 05% APS, 0.2% TEMED) was added, and then a sample comb was set. The cathode electrophoresis buffer composition was 25 mM Tris, 192 mM glycine and 0.1% SDS, and the anode electrophoresis buffer composition was 150 mM Tris-HCl (pH 8.8). The sample was mixed with a dissolved equal amount of 0.5% agarose gel, dropped into the sample well and coagulated, followed by SDS-PAGE.

  As a sample to be separated by SDS-PAGE, visible-stained SeeBlue plus 2 M.P. W. Marker (Invitrogen) and fluorescently labeled DyLight fluorescent protein molecular weight markers (PIERCE) were used.

  As a result of applying voltage at a constant current of 20 mA for 45 minutes, a clear protein band was detected even when one gel plate was a stripe electrode substrate (FIG. 7). On the other hand, it was confirmed that the protein was not moved at all on the substrate on which the entire surface of the substrate was formed of Pt, and voltage could not be applied (FIG. 8). In addition, the mobility was slower when the stripe electrode substrate was used.

  Further, in the stripe electrode substrate, gas was generated on the stripe electrode substrate during SDS-PAGE. It was inferred that the mobility of the protein was slowed as a result of the gas being peeled off from the stripe electrode substrate due to the generation of the gas and the buffer invading and depriving the buffer power. In addition, although the bubbles do not have a great influence on the degree of protein separation, a method for suppressing the generation of bubbles was examined in order to prevent uniform voltage application on the stripe electrode in subsequent transfer.

[Example 3: Examination of method for suppressing generation of bubbles]
The generation of bubbles was assumed to be caused by electrolysis of water on each electrode due to a potential difference generated between each electrode (line) of the stripe electrode. Therefore, it was predicted that the generation of bubbles could be prevented by setting the potential difference generated between the electrodes to 1 V or less, which is the water decomposition voltage.

  Therefore, the generation of bubbles in SDS-PAGE when the applied voltage was changed using a stripe electrode substrate having a Pt line width of 100 μm and a Pt line interval of 100 μm was examined. Table 1 shows the calculated voltage and the calculated value of the voltage between the electrodes at that time. The voltage between the electrodes is a quotient of the voltage applied to the gel and the number of stripes.

  As a result, bubbles are generated when the voltage between the electrodes is 0.4 V or more (gel applied voltage is 100 V or less), whereas bubbles are not generated when the voltage is 0.3 V or less (gel applied voltage is 75 V or less). Became clear. Further, when a stripe electrode substrate having a Pt line width of 50 μm and a Pt line interval of 50 μm was used, no bubbles were generated even when the gel application voltage was 150 V (the voltage between the electrodes was 0.3 V). From the above, it has been clarified that the condition for suppressing the generation of bubbles is that the voltage between the electrodes is 0.3 V or less.

  The decomposition voltage of water is affected by the electrode material and the components in the buffer. It is presumed that the applied voltage between the electrodes can be increased by using zinc or copper having a lower overvoltage than Pt.

[Example 4: Implementation with commercially available equipment]
Striped electrodes were prepared by the same procedure as described in Example 1 on a minigel-sized gel plate (10 cm × 8.2 cm, 3 mm thick, Bio-Rad). 500 stripes were prepared in a 10 cm substrate so that L / S = 80 μm / 120 μm. At this time, the potential difference between the stripe electrodes is 0.2V. Both were stripe electrode substrates, one was a stripe electrode substrate, and both were glass substrates, and the gel was polymerized to perform SDS-PAGE. SDS-PAGE was performed as a standard method using a mini-PROTEAN 3 cell (Nippon Bio-Rad Laboratories) apparatus. SDS-PAGE was performed at 100 V for 1 hour. Under these conditions, no bubbles were generated on the stripe electrode substrate. A sharp band was obtained even when one or both gel plates were striped electrode substrates, and the moving speed was not lowered.

  Since the present invention provides an improved electrophoresis technique, it can be used in the field of separation and analysis of biomaterials.

It is a schematic diagram which shows the structure of the electrophoresis apparatus which concerns on one Embodiment of this invention. It is a perspective view which shows the structure of the isoelectric focusing apparatus which concerns on one Embodiment of this invention. It is a top view which shows the structure of the isoelectric focusing apparatus which concerns on one Embodiment of this invention. It is a side view which shows the structure of the isoelectric focusing apparatus which concerns on one Embodiment of this invention. It is explanatory drawing of the electrophoresis method which concerns on one Embodiment of this invention. It is a photograph of a plurality of electric conductors concerning one embodiment of the present invention. It is a photograph which shows the result of the electrophoresis method which concerns on one Embodiment of this invention. It is a graph which shows the result of the electrophoresis method which concerns on one Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Electrophoresis apparatus 101,102 Buffer solution tank 103 Separation part 104,105 Electrode (voltage application means)
106 Stripe electrode (conductor)
120 Medium 200 Isoelectric focusing device 201 Separation tank 202, 203 Electrode (voltage applying means)
204, 205 Striped electrode (conductor)
206 Lid member 220 Medium

Claims (6)

Electricity for separating a substance to be separated in a medium disposed in contact with 250 or more linear conductors each having a width of 100 μm or less and a distance between adjacent conductors of 100 μm or less An electrophoresis apparatus,
Voltage applying means for applying a voltage to the medium;
The electrophoretic device, wherein the voltage applying means applies the voltage so that a potential difference between the adjacent conductors is greater than 0 V and less than or equal to 0.3 V.   The electrophoretic device according to claim 1, wherein the voltage applying unit applies the voltage to the conductors. The linear conductor is electrophoretic device according to claim 1 or 2 said voltage applying means is characterized by being arranged parallel to each other Cartesian direction for applying a voltage to the medium. The conductor, platinum, zinc, and electrophoretic device according to any one of claims 1 to 3, characterized in that it consists of a metal selected from the group consisting of copper. It is an isoelectric focusing apparatus, The electrophoresis apparatus as described in any one of Claims 1-4 characterized by the above-mentioned. Electricity for separating a substance to be separated in a medium disposed in contact with 250 or more linear conductors each having a width of 100 μm or less and a distance between adjacent conductors of 100 μm or less An electrophoresis method comprising:
An electrophoretic method comprising applying a voltage to the medium such that a potential difference between adjacent conductors is greater than 0V and less than or equal to 0.3V.