JP5847213B2 - Two-dimensional electrophoresis method - Google Patents

Description

  The present invention relates to a two-dimensional electrophoresis method. More specifically, the present invention relates to a two-dimensional electrophoresis method in which the gel length of the first-dimensional electrophoresis gel is set to be relatively short and the gel concentration at the proximal end in the migration direction of the second-dimensional electrophoresis gel is lowered.

  In the present invention, “gel concentration” directly means the monomer concentration during the polymerization reaction of the gel, but the higher the monomer concentration during the polymerization reaction, the denser the network structure of the gel. Means the density of the gel network.

  Conventionally, various methods for separating and purifying proteins and nucleic acids from cell extracts have been studied. Examples of precipitation and centrifugation using salt concentration are examples.

  In addition, a number of purification methods using the charge of proteins and nucleic acid residues and the difference in molecular weight have been studied. Examples of the purification method using charges include column chromatography using an ion exchange resin and isoelectric focusing. Examples of the purification method utilizing the difference in molecular weight include centrifugation, column chromatography using a molecular weight sieve, and SDS-PAGE.

  In recent years, as a method for separating and purifying various proteins from a small amount of sample, a two-dimensional electrophoresis method in which isoelectric focusing is performed in the first dimension and SDS-PAGE is performed in the second dimension is used.

JP-T-2002-503813. This patent document 1 discloses two-dimensional electrophoresis performed on the serum or plasma of a subject for diagnosis of hepatocellular cancer.

David R. M. Graham et al. “Improvements in two-dimensional gel electrophoresis by utilizing a low cost“ in-house ”neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system” Proteomics 2005, 5, 2309-2314. This non-patent document 1 discloses a certain improvement called “in-house system” in two-dimensional electrophoresis including SDS-PAGE.

  Usually, in electrophoresis, firstly, the gel length of the gel for electrophoresis corresponds to the resolution, so that the gel length is set long from the viewpoint of obtaining many spots. For example, in Patent Document 1 and Non-Patent Document 1, the gel length is 18 cm or longer in the first-dimensional isoelectric focusing of the example. Second, the gel concentration of the second-dimensional electrophoresis gel is generally set to about 12%, but this gel concentration is the gel length of the first-dimensional electrophoresis gel. Is not necessarily fully considered in relation to.

  However, with regard to the first point, the separation performance improves as the migration distance increases, but the migration time also increases and the number of migrations (throughput) per fixed time decreases. In addition, the detected spot tends to be broad. Spot broadening leads to the problem of an increase in the detection limit of substances to be separated such as proteins.

  On the other hand, when the gel length of the first-dimensional electrophoresis gel is simply set short, the migration time is shortened, the number of migrations (throughput) per fixed time is improved, and the spot in the first-dimensional electrophoresis becomes sharper. The distance between protein spots in the first-dimensional electrophoresis gel becomes compact, and the protein concentration in the spot also increases.

  Therefore, it becomes relatively difficult to transfer the protein concentrated in the compact spot of the first-dimensional electrophoresis gel to the second-dimensional electrophoresis gel, the protein migration leak becomes remarkable, and the protein spot becomes 2 There was a problem that the second-order electrophoresis broadens in the horizontal direction with respect to the migration direction.

  Therefore, the present invention ensures advantages such as shortening the migration time by setting the gel length of the first dimension electrophoresis gel to be relatively short, increasing the throughput of the first dimension electrophoresis, suppressing spot broadening, and the like. It is a problem to be solved to suppress problems such as protein migration leakage and lateral broadening of protein spots in second-dimensional electrophoresis.

(First invention)
The configuration of the first invention of the present application for solving the above-mentioned problem is that the gel length in the longitudinal direction of the first-dimensional electrophoresis gel is 5 to 10 cm, and the gel concentration at the proximal end in the migration direction of the second-dimensional electrophoresis gel This is a two-dimensional electrophoresis method in which is 3 to 6%.

(Second invention)
The configuration of the second invention of the present application for solving the above problem is a two-dimensional electrophoresis method according to the first invention, wherein the second-dimensional electrophoresis gel is a polyacrylamide gel (PAG).

(Third invention)
The configuration of the third invention of the present application for solving the above problem is a two-dimensional electrophoresis method in which the electrophoresis performed using the second-dimensional electrophoresis gel in the first invention or the second invention is SDS-PAGE. is there.

(Fourth invention)
The structure of the fourth invention of the present application for solving the above-described problem is that, in any one of the first to third inventions, the gel concentration of the tip side in the migration direction of the second-dimensional electrophoresis gel is set high. This is a two-dimensional electrophoresis method.

(First invention)
The inventor of the present application is that the gel for electrophoresis has a network structure, and the gel concentration means the density of the network structure of the gel. The concentration was considered to be a barrier when the protein was transferred from the first-dimensional electrophoresis gel to the second-dimensional electrophoresis gel. Second, the migration of proteins to the second-dimensional electrophoresis gel is defined by the relative barrier properties of this barrier, the mutual spacing of protein spots to be migrated, and the protein concentration in the spots. Thought.

  That is, if the gel length of the first-dimensional electrophoresis gel is sufficiently long (for example, 18 cm), apart from the above-described problems, the mutual distance between the protein spots to be transferred to the second-dimensional electrophoresis gel is Because it is sufficiently large and the protein concentration in the spot is sufficiently low, even if the gel concentration of the second-dimensional electrophoresis gel is as high as about 12% (high barrier property), it causes a relatively hindrance to protein migration. It is not considered.

  However, when the gel length of the first-dimensional electrophoresis gel is set to be short for the purpose of shortening the electrophoresis time in the first-dimensional electrophoresis and suppressing spot broadening, the protein smoothes the barrier of the above high barrier property. Inability to pass through, the above-described problems such as protein migration leakage and lateral broadening of protein spots occur.

  According to the study of the present inventor, when the gel length in the longitudinal direction of the first-dimensional electrophoresis gel is 5 to 10 cm in a normal specimen, at least the proximal end portion in the migration direction in the second-dimensional electrophoresis gel By setting the gel concentration to a low level in the range of 3 to 6%, the protein can smoothly pass through the barrier of this low barrier property, and problems such as protein migration leakage and lateral broadening of the protein spot can be suppressed. .

  Therefore, according to the first invention, the gel length of the first-dimensional electrophoresis gel is set to be relatively short, and the advantages such as shortening of the migration time, high throughput of the first-dimensional electrophoresis, and suppression of spot broadening are ensured. At the same time, it is possible to suppress problems such as protein migration leakage and accompanying lateral broadening of protein spots in the second-dimensional electrophoresis.

(Second invention)
The type of the second-dimensional electrophoresis gel used in the present invention is not necessarily limited, but a polyacrylamide gel (PAG) can be preferably exemplified.

(Third invention)
In the two-dimensional electrophoresis method of the present invention, the type of second-dimensional electrophoresis is not necessarily limited, but SDS-PAGE can be preferably exemplified.

(Fourth invention)
In the two-dimensional electrophoresis method of the present invention, if necessary (for example, when it is desired to increase the resolution in the second-dimensional electrophoresis), the gel on the tip side in the migration direction of the second-dimensional electrophoresis gel This can be dealt with by setting the density high.

The result of the two-dimensional electrophoresis which concerns on the comparative example 1 with respect to 1st Example and 1st Example is shown.

It is a schematic diagram which shows the gel length of each gel of the two-dimensional electrophoresis which concerns on 1st Example, and the pH gradient or gel concentration gradient of those gels.

It is a schematic diagram which shows the gel length of each gel of the two-dimensional electrophoresis which concerns on the comparative example 1 with respect to a 1st Example, and the pH gradient or gel concentration gradient of those gels.

The result of the two-dimensional electrophoresis which concerns on the comparative example 2 with respect to a 1st Example is shown.

It is a schematic diagram which shows the gel length of each gel of the two-dimensional electrophoresis which concerns on the comparative example 2 with respect to a 1st Example, and the pH gradient or gel concentration gradient of those gels.

  Next, modes for carrying out the present invention will be described including the best mode.

[First dimension electrophoresis]
The first-dimensional electrophoresis of the present invention is not particularly limited, but isoelectric focusing can be preferably exemplified. When isoelectric focusing is performed in the first dimension, separation is performed using the isoelectric point of the substance to be separated such as protein in the sample. The positively charged separation target substance moves to the cathode side, while the negatively charged separation target substance moves to the anode side. Then, at the position of the gel having a pH equal to the isoelectric point (pI), the net charge of the substance to be separated becomes zero, and the migration is stopped. Therefore, since the charged compound moves after the start of electrophoresis, a current flows.

[1D electrophoretic gel]
The first-dimensional electrophoresis gel is not particularly limited, but an isoelectric focusing gel can be preferably exemplified. As a kind of gel, a polyacrylamide gel can be illustrated preferably. The form of the first-dimensional electrophoresis gel is not particularly limited as long as it can be provided on the sample application side (electrophoresis direction proximal end side) of the second-dimensional electrophoresis gel. A rod shape and a columnar shape can be exemplified as preferred forms.

  In the present invention, the “longitudinal direction of the first-dimensional electrophoresis gel” generally refers to the axial direction of a rod-shaped or cylindrical gel, and more essentially refers to the electrophoresis direction of the analyte to be separated. means. The gel length in the longitudinal direction of the first-dimensional electrophoresis gel is preferably 5 to 10 cm, particularly preferably 5 to 8 cm.

  When the first dimensional electrophoresis is isoelectric focusing, it is desirable to set an appropriate pH gradient in the isoelectric focusing gel. The pH gradient can be appropriately set according to the type of specimen and the preparation method. As the setting method, an amphoteric carrier is added to the gel and an electric field is applied to form a desired pH gradient, and a monomer derivative having various isoelectric side chains is used simultaneously with the preparation of the polymer gel. A method (IPG method) or the like for forming a pH gradient in a fixed manner is preferably used.

  For example, when the specimen is an extract of a biological cell such as a human cell, the first dimensional electrophoresis is preferably isoelectric focusing, and the gel used for the isoelectric focusing has a pH range of the gel. “A <b” and “b> when the gel pH gradient is 3 to 10 and the gel length up to pH 5 is a, the gel length of pH 5 to 7 is b, and the gel length of pH 7 or more is c. c ", and more preferably, when the total length of the gel is 1, a is in the range of 0.15 to 0.3, b is in the range of 0.4 to 0.7, c is in the range of 0.15 to 0.3, and particularly preferably satisfies the relationship of “a + c ≦ b”.

  The inventor of the present application has empirically grasped that specimens prepared from extracts of biological cells such as human cells often contain many proteins having an isoelectric point at pH 5-7. The gel used for the isoelectric focusing can separate a large number of proteins having an isoelectric point at pH 5 to 7 with high resolution. On the other hand, proteins having isoelectric points in other pH ranges are less than proteins having isoelectric points at pH 5 to 7 (the number of separation targets is small). Even if the gradient is steep compared to pH 5-7, sufficient separation ability can be exhibited. The gel length can be suppressed by providing the necessary resolution in each pH range. On the other hand, since the migration distance of the protein to be separated is reduced, a high throughput can be realized. In addition, since the pH gradient is steep in the other pH ranges, the protein to be separated becomes a high concentration spot. Therefore, the detection limit value of the protein to be separated having an isoelectric point in the other pH range can be lowered.

[Isoelectric focusing method]
When the first-dimensional electrophoresis is isoelectric focusing, the equipment used for the electrophoresis is not particularly limited. However, in order to realize a small apparatus, high resolution, and high throughput, an electrophoresis apparatus that matches the use of a gel having a gel length of 5 to 10 cm is preferable.

  The protocol for the first dimension electrophoresis is not particularly limited. However, in order to realize high resolution and high throughput, it is necessary to pay attention to the electrophoresis protocol. When the first-dimensional electrophoresis is isoelectric focusing, it is preferable to remove as much as possible of coarse substances that are charged substances that are not subject to separation / purification in the stage of preparing the sample solution. For example, when the target of separation / purification is a protein, phospholipids, nucleic acids including genomic DNA and RNA, fatty acids, metal ions, surfactants for extraction, and the like are included in the rough matter. However, since a small amount of the rough matter may remain in the sample, it is preferable to remove the sample without applying a large load to the instrument in isoelectric focusing. The coarse substance has a high moving speed in the gel. Therefore, in the early stage of the isoelectric focusing protocol, a constant voltage step is performed by applying a constant voltage having a value within a range of 100 V to 600 V, which is a relatively weak voltage for each gel containing the specimen, and the electrophoresis is performed for 30 minutes. It is preferable to start a voltage increasing step for increasing the voltage from the constant voltage after the per-current change width is in the range of 5 μA, and to make the final voltage in the voltage increasing step within a range of 3000V to 6000V. If a high voltage is used in the early stage of the isoelectric focusing protocol, the impurities move rapidly to the electrode side, causing a strong current to flow. There is a risk that it will worsen (a clogging of spots in the gel will occur). Further, it is preferable to keep the gel temperature constant during electrophoresis so that the isoelectric point of the substance to be separated does not shift.

  According to the above embodiment, the following effects can be expected. That is, by performing the constant voltage process at a low constant voltage of 100 V to 600 V before the voltage increasing process, the positively charged rough matter is quickly moved to the cathode, and the negatively charged rough matter is quickly moved to the anode. As a result, it is possible to remove coarse substances from the gel without imposing a load on the separation target substance in the device or specimen. Further, since it is possible to determine the removal of the rough matter by measuring the current change per unit time, the constant voltage process is not insufficient and the constant voltage process is not too long. Furthermore, by setting the final voltage to a high value of 3000 V to 6000 V, a high Vhr value can be obtained in a shorter electrophoresis time, and high throughput of isoelectric focusing can be realized.

  The mode of voltage increase in the voltage increase step is not particularly limited, but it is preferable to gradually increase the voltage. Specifically, the upper limit of the current value of the electrophoresis apparatus is set to a value within the range of 40 to 80 μA per gel. Then, it is preferable to increase the voltage to the final voltage so that the gel temperature is kept constant.

[Second dimension electrophoresis]
The second-dimensional electrophoresis of the present invention is not particularly limited, but SDS-PAGE can be preferably exemplified. In SDS-PAGE, SDS (sodium dodecyl sulfate) as a surfactant is added to a sample to solve the higher-order structure of the protein contained in the sample, and the amino acid residue charge of the protein is also relatively decreased by SDS. At the same time, electrophoresis is performed using the molecular sieve effect.

[Second-dimensional SDS-PAGE]
The second-dimensional electrophoresis gel is not particularly limited, but an SDS-PAGE gel can be preferably exemplified. Although the kind of gel is not specifically limited, A polyacrylamide gel can be illustrated preferably. In this case, the gel concentration indicates the acrylamide concentration.

  In the process of setting the first-dimensional electrophoresis gel on the second-dimensional electrophoresis gel after the completion of the first-dimensional electrophoresis, the gelation temperature is 35 to 40 ° C. as an agarose for adhesion (encapsulation). It is preferable to use a melting point agarose and install the first-dimensional electrophoresis gel after pouring the adhesion agarose onto the second-dimensional electrophoresis gel in advance.

  According to the above embodiment, the gelation of the agarose for adhesion is prevented from being weakened (the gel is loosened) by the heat generated during the second-dimensional electrophoresis. Accordingly, it is possible to suppress problems such as detection spot spread, detection limit increase, and detection protein decrease due to such problems in the second-dimensional electrophoresis. In addition, since the high melting point agarose comes into contact with the gel for the second dimensional electrophoresis by pre-inserting the adhesive agarose, it is rapidly cooled, so even when urea is added to the SDS equilibration buffer, the thermal decomposition is not caused. Hard to happen.

  The apparatus which performs SDS-PAGE is not specifically limited. Moreover, regarding the PAG (polyacrylamide gel) for performing SDS-PAGE, the total concentration (T%) of acrylamide and the crosslinking agent is preferably 3-20. Moreover, it is preferable that the ratio (C%) which a crosslinking agent accounts in the total weight of acrylamide and a crosslinking agent is 0.05-2.0.

[Gel concentration at the base of the gel for the second dimensional electrophoresis]
Since the gel length of the first-dimensional electrophoresis gel is set to be short, in SDS-PAGE performed as the second dimension, the gel concentration at the base end in the migration direction in the electrophoresis gel is as low as about 3 to 6%. The concentration is preferred.

  According to the above embodiment, the following effects can be expected. That is, if the gel length of the first-dimension isoelectric focusing gel is shortened to about 5 to 10 cm, for example, the first-dimension electrophoresis time can be shortened and high throughput can be achieved. The distance between each other becomes compact, and the protein concentration in the spot increases. On the other hand, if the gel concentration at the base end of the migration direction of the gel for the second dimension electrophoresis is high (the gel network is dense), the protein concentrated in the spot is transferred to the gel for the second dimension electrophoresis. High barrier property against migration, protein migration leakage becomes remarkable, and spots broaden laterally with respect to the migration direction. Such an inconvenience is suppressed by the above embodiment.

  The change in gel concentration in the migration direction of the second-dimensional electrophoresis gel is not particularly limited, but from the viewpoint of suppressing spot broadening and lowering the detection limit of the sample, the concentration of the gel toward the sample migration direction is reduced. It is preferable to increase linearly continuously. It is preferable that the gel concentration at the tip side in the migration direction is in the range of 10 to 20%, particularly in the range of 10 to 15%.

  For example, in a polyacrylamide gel, a concentration gradient gel can be prepared by mixing two types of acrylamide solutions and adding a gradient of acrylamide concentration. If a spot was newly detected in the second-dimensional electrophoresis performed on the gel with the gel concentration gradient, it was not detected in the second-dimensional electrophoresis performed on the gel without the gel concentration change. This means that the detection limit value for the newly detected spot in the electrophoresis is lowered.

[Sample preparation]
The specimen applied to the two-dimensional electrophoresis method of the present invention is not particularly limited, but various samples including extracts derived from animals, plants, microorganisms, chemically and biochemically synthesized compounds, proteins, nucleic acids and the like. Samples can be applied. The specimen is preferably an extract of a biological cell, in particular an animal cell, in particular a human cell.

  The above-mentioned gel pH gradient is set by, for example, isoelectric point distributions of various proteins contained in biological cell extracts concentrated relatively in the pH 5-7 region in both types and amounts of proteins. The gel length can be shortened without substantially impairing the high resolution.

  In the gel for electrophoresis, the speed of electrophoresis varies depending on the molecular weight, but a substance having a low molecular weight such as sodium ion does not pass through the sieve and moves quickly in the gel. In addition, genomic DNA has a large molecular weight, but since it is highly negatively charged, it moves quickly to the anode. In sample preparation, it is preferable to remove impurities that are not subject to separation / purification in order to reduce the load on the instrument, shorten the constant voltage step, and suppress clogging of spots in the gel. For this purpose, various pretreatments such as dialysis, precipitation, centrifugation, chromatography, and fractionation utilizing hydrophilic-hydrophobic interaction can be applied. When proteins are to be separated and purified, precipitation with an acid and precipitation with an organic solvent can be preferably exemplified. Precipitation with TCA (trichloroacetic acid) and precipitation with acetone can be exemplified as further preferred methods.

  A specimen to be subjected to separation / purification can be dissolved in a swelling buffer solution for gel used for isoelectric focusing, to obtain a swelling specimen solution, and the specimen can be taken into the gel as the gel swells. Alternatively, the specimen can be dissolved in an appropriate solution and applied to the gel after swelling.

  In the preparation of such a swelling gel for isoelectric focusing, oil is poured into the gel after applying the swelling sample solution to the entire gel. A method in which the oil component is poured from the side end portions in the longitudinal direction of the gel, particularly from the side end portions on both sides in the longitudinal direction of the gel, instead of pouring the oil component is particularly preferable. As the oil component, silicone oil or mineral oil, particularly the former is preferred.

  According to the above embodiment, the oily component spreads from the side end portion of the gel toward the center portion and covers the gel. If the oil component covers the gel for a while, the specimen is efficiently taken into the gel. At this time, since the swelling sample solution is repelled by the oily component spreading from the side end portion of the gel toward the center portion, the penetration of the swelling sample solution into the gel is promoted, and the penetration of the sample into the entire gel is quick and easy. Complete well. When an oily component is poured into the gel surface as in the past, the oily component spreads from the gel, so that part of the unstained sample solution for swelling that is pushed by the flow diffuses from the gel. Although it is thought that this has led to a decrease in detectable proteins and the like and insufficient swelling of the gel, according to the above-described embodiment, such drop-out of the analyte component does not occur.

  Before shifting to the second-dimensional electrophoresis, the first-dimensional electrophoresis gel that has finished the first-dimensional electrophoresis is equilibrated with a mobile phase or the like used for the second-dimensional electrophoresis. After equilibration, the first-dimensional electrophoresis gel is brought into close contact with the specimen application side of the second-dimensional electrophoresis gel using a gel support material such as agarose. Thereafter, the second-dimensional electrophoresis is started.

  When the second dimensional electrophoresis is SDS-PAGE, electrophoresis is performed using a molecular weight sieve, so the first dimensional electrophoresis gel is first equilibrated with a buffer containing a reducing agent in order to solve the three-dimensional structure of the specimen. It is preferable to do. Although the said reducing agent is not specifically limited, DTT can be illustrated preferably. Thereafter, in the second equilibration of the first-dimensional electrophoresis gel, it is preferable to equilibrate with a buffer containing an alkylating agent. Although an alkylating agent is not specifically limited, Iodoacetamide can be illustrated preferably.

  Examples of the present invention and comparative examples will be described below. The technical scope of the present invention is not limited by these examples and comparative examples.

[First embodiment]
(Extraction of protein)
A mammalian cell lysis kit, which is a protein extract, from one culture (about 1 cm ² ) of reconstituted three-dimensional cultured skin consisting of human keratinocytes (trade name LabCyte EPI-MODEL 12 manufactured by Japan Tissue Engineering Co., Ltd.); It was immersed in 500 μl of MCL1 (manufactured by SIGMA-ALDRICH) and crushed by shaking using voltex at 4 ° C. for 2 hours. After this shaking crushing, the protein extract was recovered. The composition of the above mammalian cell lysis kit; MCL1 is as follows.
50 mM Tris-HCl pH 7.5
1 mM EDTA
250 mM NaCl
0.1% (w / v) SDS
0.5% (w / v) Deoxycholic acid sodium salt
1% (v / v) Igepal CA-630 (SIGMA-ALDRICH surfactant (Octylphenoxy) polyethoxyethanol)
Appropriate amount of Protease Inhibitor
Then, precipitation operation was performed twice using a 2D-CleanUP kit [manufactured by GE Healthcare Bioscience Co., Ltd. (hereinafter abbreviated as GE)]. In the first precipitation operation, TCA was added to the recovered protein extract to perform precipitation, and the precipitate generated by the operation (TCA precipitation) was recovered. In the second precipitation operation, acetone was added to the recovered TCA precipitate to perform precipitation, and the precipitate (specimen) obtained by the operation was recovered. The collected sample was a total amount of 500 μg.

(Preparation of sample solution)
A 30 μg portion of the obtained specimen is dissolved in 130 μl of DeStreak Rehydration Solution (GE), which is a buffer for swelling the gel for first-dimensional isoelectric focusing, and the specimen for first-dimensional isoelectric focusing is dissolved. A solution (specimen solution for swelling) was used. The composition of DeStreak Rehydration Solution is as follows.
7M Thiorea
2M Urea
4% (w / v) CHAPS:
3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate
0.5% (v / v) IPGbuffer; appropriate amount of DeStreak Reagent manufactured by GE; appropriate amount of BPB (bromophenol blue) manufactured by GE
(Preparation of gel for 1D isoelectric focusing)
A first-dimensional isoelectric focusing gel (polyacrylamide gel) used in this example was prepared by the IPG method described above. This gel was a rod-like gel having a length of 7 cm and a diameter of 0.3 cm, T = 4%, C = 3%, and the pH range was set to 3-10.

(Penetration of specimen into gel for 1D isoelectric focusing)
After immersing the above-mentioned first-dimensional isoelectric focusing gel in 130 μl of the first-dimensional isoelectric focusing sample solution (swelling sample solution), silicon oil is poured, and the silicon oil covers the gel. In this state, the sample solution was allowed to penetrate the gel overnight at room temperature. Thereafter, the silicon oil was discarded.

(First-dimensional isoelectric focusing)
In this example, IPGphor manufactured by GE and Cup Loading Manifold Light Kit were used as the electrophoresis apparatus.

  A filter paper moistened with water was provided at both ends of the gel infiltrated with the specimen, and the electrode was set with the filter paper sandwiched between the gel and the gel. Thereafter, the entire gel and filter paper were immersed in silicon oil.

  The upper limit of the current value of the isoelectric focusing device is set to 75 μA per gel, and the voltage program is set to (1) a constant voltage step to 300 V constant voltage to 750 Vhr (30 minutes before the end of the step) (2) The voltage was gradually increased to 1000 V over 300 Vhr), (3) the voltage was gradually increased to 5000 V over 4500 Vhr, and (4) the voltage was fixed at 5000 V thereafter. The first dimension isoelectric focusing was performed until the total Vhr was 12000 in terms of voltage.

(SDS equilibration of isoelectric focusing gel)
After performing the first-dimension isoelectric focusing, remove the gel from the isoelectric focusing device, immerse the gel in an equilibration buffer containing a reducing agent, and shake at room temperature for 15 minutes. That ’s it. The composition of the equilibration buffer containing the reducing agent is as follows.
100 mM Tris-HCl (pH 8.0)
6M Urea
30% (v / v) Glycerol
2% (w / v) SDS
1% (w / v) DTT
Next, the equilibration buffer containing the reducing agent was removed, the gel was immersed in an equilibration buffer containing an alkylating agent, and shaken at room temperature for 15 minutes to obtain an SDS equilibrated gel. The composition of the equilibration buffer containing the alkylating agent is as follows.
100 mM Tris-HCl (pH 8.0)
6M Urea
30% (v / v) Glycerol
2% (w / v) SDS
2.5% (w / v) Iodoacetamide
(Second-dimensional SDS-PAGE)
In this example, XCell SureLock Mini-Cell manufactured by Invitrogen was used as an electrophoresis apparatus. As the gel for the second dimensional electrophoresis, NuPAGE 4-12% Bis-Tris Gels manufactured by Invitrogen (the gel length in the specimen application direction was 8 cm and the gel length in the migration direction was 8 cm) was used.
In addition, an electrophoresis buffer having the following composition was prepared and used.
50 mM MOPS
50 mM Tris base
0.1% (w / v) SDS
1 mM EDTA
In this example, 0.5% (w / v) agarose S (manufactured by Nippon Gene: melting temperature ≦ 90 ° C., so-called high melting point agarose having a gelation temperature of 37 ° C. to 39 ° C.) And an agarose solution for adhesion in which an appropriate amount of BPB (bromophenol blue) was dissolved.

  After the SDS-PAGE well was thoroughly washed with the above-mentioned electrophoresis buffer, the buffer used for the washing was removed. Next, an agarose solution for adhesion sufficiently dissolved in the well was added. Next, the SDS-equilibrated gel was immersed in agarose, and the SDS-equilibrated gel and tweezers gel were brought into close contact with each other. After confirming that the agarose was sufficiently hardened with the two gels in close contact, electrophoresis was performed at a constant voltage of 200 V for about 45 minutes.

(Fluorescent staining of gel)
The gel was fluorescently stained using SyproRuby (Invitrogen).

  First, the tapper to be used was thoroughly washed with high-concentration ethanol in advance. Remove the gel for the second dimensional electrophoresis after electrophoresis from the SDS-PAGE instrument, place it on a washed tapper, and immerse it in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. We went twice. Thereafter, the aqueous solution was replaced with water and immersed for 10 minutes. Next, the 2D gel was immersed in 40 cc SyproRuby (Invitrogen) and shaken overnight at room temperature. Next, SyproRuby was removed, and the gel for 2D electrophoresis was washed with water, and then shaken with an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. Further, the aqueous solution was replaced with water and shaken for 30 minutes or more.

(analysis)
The gel for two-dimensional eye electrophoresis subjected to the above-described series of treatments was subjected to fluorescence image scanning using Typhoon 9400 (manufactured by GE). The result of two-dimensional electrophoresis is shown in FIG. The left end is a marker used in SDS-PAGE.

[Second Embodiment]
In the second example, 2D-DIGE was performed. In the second embodiment, the procedure in the section “(Sample solution preparation)” in the procedure described in the first embodiment is changed to the procedure in the following “(Sample solution preparation in 2D-DIGE)” section. In addition, the same procedure as in the first example was performed except that the process of “(fluorescence staining of gel)” was omitted.

(Preparation of specimen solution in 2D-DIGE)
The total amount of the obtained specimen was dissolved in 100 μl of a solution having the following composition.
30 mM Tris-HCl (pH 8.5)
2M ThioUrea
7M Urea
4% (w / v) CHAPS
160 pmol of Cydye (manufactured by GE) was added to 20 μg of the dissolved sample, and the container containing the solution was allowed to stand on ice for 30 minutes. Thereafter, 0.5 μl of a 10 mM lysine aqueous solution was added, and the container was allowed to stand on ice for another 10 minutes. After such treatment, the solution was diluted with DeStreak Rehydration Solution to a volume of 130 μl suitable for isoelectric focusing. After the measurement, the sample was sufficiently stirred and allowed to stand on ice for 10 minutes or more to prepare a sample solution for first-dimension isoelectric focusing.

[Comparative Example 1 for First Example]
In this comparative example, except for the following 1) to 3), all steps from sample preparation to fluorescent staining and analysis of the gel were performed in the same manner as in the first example.
1) The total amount of the sample dissolved in 340 μl of DeStreak Rehydration Solution (manufactured by GE), which is a buffer solution for swelling the first-dimensional isoelectric focusing gel, was 270 μg.
2) The gel length in the longitudinal direction of the first-dimensional isoelectric focusing gel was 18 cm, the migration distance was increased, and isoelectric focusing was performed according to the following protocol.
Equipment used: Cool Holester (registered trademark) IPG-IEF Type-PX (Anatech Corporation)
In the voltage program, (1) as a constant voltage step, electrophoresis was performed at a constant voltage of 500 V up to 1000 Vhr (the change in the current amount for 30 minutes before the end of the step was within 5 μA), and (2) 3000 V over 10700 Vhr. The voltage was gradually increased to (3), and then the total Vhr was 46700 Vhr at a constant voltage of 3500 V, and the first-dimension isoelectric focusing was performed. In the isoelectric focusing in this comparative example, no upper limit is set for the current value.
3) The gel length in the sample application direction of the second dimension gel was 20 cm, the gel length in the migration direction was 20 cm, the acrylamide concentration in the migration direction was fixed at 12%, and the electrophoresis was performed at a constant current of 30 mA for 3 hours.
Equipment used: Cool Holester (registered trademark) SDS-PAGE Dual-200 (manufactured by Anatec Corporation).

  The result of two-dimensional electrophoresis in this comparative example is shown in FIG. In FIG. 1 (a) and FIG. 1 (b), the square regions corresponding to each other by solid arrows in the figures correspond to each other in molecular weight. In comparison between FIG. 1 (a) and FIG. 1 (b), the spot seen in FIG. 1 (b) as a comparative example is also in the first-dimensional migration direction as compared with FIG. 1 (a) as an example. It is also broad overall in the direction of migration in the second dimension. Further, in this comparative example, the amount of the sample subjected to electrophoresis is 270 μg, which is 9 times that of the first embodiment, but the number of spots that can be detected is shown in FIG. 1A, which is the result of the first embodiment. There are few compared. Furthermore, the total migration time for the two-dimensional electrophoresis of this comparative example was 22 hours, and the total of 7.5 hours in the first example. Therefore, it can be said that the first example is a two-dimensional electrophoresis method that realizes high throughput, broad suppression, and low detection limit (very little protein migration leakage).

[Comparative Example 2 with respect to the first embodiment]
In this comparative example, except for the following changes, all steps from specimen preparation to gel fluorescence staining and analysis were performed in the same manner as in the first example.

1) Concentration gradient in the migration direction of the second-dimensional electrophoresis gel Gel concentration at the proximal end in the migration direction: 10%
Gel concentration at the tip of the migration direction: 20%
(A linear concentration gradient is provided from the base end of the migration direction to the tip of the migration direction)
2) The contrast (brightness) in the analysis was set higher than in the first example.

  The results of two-dimensional electrophoresis in this comparative example are shown in FIG. The left end of FIG. 4 is a marker used in SDS-PAGE.

  In the portion surrounded by a solid line and a substantially circular shape shown in the upper left portion of FIG. 4, the spot broadening is remarkable as compared with the upper left portion of FIG. When the gel concentration as high as 10% acts as a barrier, it can be confirmed that the spots spread left and right.

  The composition of the marker used in the first example and this comparative example is the same, and the amount of the marker used is the same. However, in this comparative example, the contrast in the analysis is set higher than in the first example, so the darkness of the marker at the left end of the gel is as shown in FIG. The one is darker. Comparing in consideration of such differences in marker darkness, it can be determined that the spots in the gel in FIG. 4 are thinner. That is, the amount of protein detected in this comparative example is smaller than that in the first example. In this comparative example, it was also confirmed that a high gel concentration of 10% acts as a barrier, so that there are many leakages of protein migration to the second-dimensional electrophoresis gel.

  Next, after unifying the spot detection conditions in the analysis, the number of spots in each gel detected using the image analysis system ImageMaster 2D Platinum (manufactured by GE) is shown in FIG. About 1500 spots from the gel, about 600 spots from the gel shown in FIG. 1 (b), and about 1000 spots from the gel shown in FIG. That is, about 500 spots are not detected in this comparative example compared to the first embodiment. The reason for this is that the horizontal spread of the spots occurred due to the high barrier property at the base end of the migration direction of the second-dimensional electrophoresis gel (the spots were bound to each other and / or below the detection limit value). It was thought that the protein concentration was lower than the detection limit value due to protein migration leakage.

  According to the present invention, the gel length of the first-dimensional electrophoresis gel is set to be relatively short, shortening the migration time, ensuring advantages such as higher throughput of the first-dimensional electrophoresis, suppression of spot broadening, and the associated protein Migration defects such as lateral broadening of protein spots in two-dimensional electrophoresis can be suppressed.

1,3,5 First-dimensional isoelectric focusing gel 2,4,6 Second-dimensional SDS-PAGE gel

Claims (4)

The first dimension isoelectric focusing that satisfies the following condition (1), the equilibration buffer treatment that satisfies the following condition (2), and the two-dimensional condition that satisfies the following condition (3): Performing SDS-PAGE of the eye,
Each of these steps is performed in the order of first-dimensional isoelectric focusing, equilibration buffer treatment, and second-dimensional SDS-PAGE.
(1) As the first-dimensional electrophoresis gel, the gel length in the longitudinal direction is 5 to 10 cm, and the first-dimensional electrophoresis gel has a flat rod-like form that can be provided on the specimen application side of the second-dimensional electrophoresis gel. Use.
(2) The gel subjected to the isoelectric focusing is equilibrated with a buffer containing a reducing agent and equilibrated with a buffer containing an alkylating agent.
(3) Bis-Tris Gel which is a second-dimensional electrophoresis gel having a concentration gradient in which the gel concentration at the base end in the migration direction is 3 to 6% and the gel concentration at the tip side in the migration direction is 10 to 20%. Is used in a state in which the first-dimensional electrophoresis gel subjected to the equilibration buffer treatment is closely attached to the specimen application side . 2. The two-dimensional electrophoresis method according to claim 1, wherein the specimen contains a protein. 2. The two-dimensional electrophoresis method according to claim 1, wherein the specimen is an extract derived from an animal, a plant, or a microorganism and contains a protein. The two-dimensional electrophoresis method according to claim 2, further comprising performing precipitation with an acid and precipitation with an organic solvent in a specimen preparation stage of the two-dimensional electrophoresis method.