JP5513802B2 - Isoelectric focusing gel and isoelectric focusing method - Google Patents

Description

  The present invention relates to an isoelectric focusing gel and an isoelectric focusing method. More specifically, the present invention relates to setting a characteristic pH gradient in a gel used for isoelectric focusing. The present invention is preferably applied not only to isoelectric focusing performed alone but also to isoelectric focusing performed as first-dimensional electrophoresis in two-dimensional electrophoresis.

  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 dodecylsulfate-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.

  However, in Patent Document 1 and Non-Patent Document 1 described above, the first-dimensional isoelectric focusing is performed using a large gel having a gel length of 18 cm or more. Usually, in electrophoresis, the longer the gel length used for electrophoresis, the better the resolution. Therefore, the separation and purification of the sample becomes more reliable. On the other hand, the longer the gel length, the longer the sample travel distance, and the longer the time required for electrophoresis. For this reason, there is a problem in that the number of migrations (throughput) per fixed time decreases. Therefore, particularly in the comprehensive analysis of proteins and nucleic acids, an electrophoretic method with high resolution and high throughput using a small apparatus (short gel) is desired.

  Therefore, the present invention should solve the problem of shortening the gel length of the gel for isoelectric focusing without impairing the high resolution, thereby enabling downsizing of the electrophoresis apparatus, shortening of the electrophoresis time, and high throughput. Let it be an issue.

(First invention)
The configuration of the first invention of the present application for solving the above problems is that the gel length is in the range of 5 to 10 cm, the pH range of the gel is 3 to 10, and the pH gradient of the gel with respect to the migration direction is up to pH 5. The gel for isoelectric focusing satisfies the relationship of “a <b” and “b> c”, where a is the gel length of b, b is the gel length of pH 5-7, and c is the gel length of pH 7 or higher. It is.

(Second invention)
The configuration of the second invention of the present application for solving the above problem is that the pH gradient with respect to the migration direction of the gel for isoelectric focusing according to the first invention is 0.15 when the total length of the gel is 1. It is a gel for isoelectric focusing, wherein b is in the range of 0.4 to 0.7, and c is in the range of 0.15 to 0.3.

(Third invention)
The configuration of the third invention of the present application for solving the above problem is an isoelectric focusing method in which isoelectric focusing is performed using the gel for isoelectric focusing described in the first or second invention. is there.

(Fourth invention)
The configuration of the fourth invention of the present application for solving the above problem is an isoelectric focusing method in which the isoelectric focusing method according to the third invention is performed as the first-dimensional electrophoresis in the two-dimensional electrophoresis. .

(Fifth invention)
The configuration of the fifth invention of the present application for solving the above problem is an isoelectric focusing method in which the specimen of the isoelectric focusing method according to the third or fourth invention is an extract of a biological cell. .

(Effects of the first to third inventions)
In an isoelectric focusing gel, on the premise that a constant linear pH gradient is set with respect to the migration direction, in order to increase the resolution, the gel length is generally increased as a whole. It is possible to do. However, in this case, the migration time becomes long, and it is difficult to reduce the size of the apparatus and achieve high throughput.

  On the other hand, the isoelectric points of various proteins in a sample subjected to isoelectric focusing are not necessarily distributed evenly over a wide pH range. For example, when the isoelectric points of these proteins are concentrated to some extent within a specific pH range, increasing the overall gel length unnecessarily increases the gel length in a pH range other than the specific pH range. Will be.

  In such a case, if the pH gradient of the gel is made gentler than other pH ranges only in a specific pH range, it becomes possible to efficiently separate proteins having an isoelectric point in the specific pH range, In addition, the overall gel length can be shortened as much as possible to miniaturize the electrophoresis apparatus, and high throughput can be achieved based on shortening the migration time.

  According to the results of various studies conducted by the inventors of the present application, specimens extracted from biological cells such as human cells contain various proteins whose isoelectric points are distributed in the range of pH 3 to 10, and pH 5 to 7 Proteins having an isoelectric point in the region have more types and amounts than proteins having an isoelectric point in other pH regions. Accordingly, the gel for isoelectric focusing of the first invention has a reasonably shortened gel length while maintaining a high resolution, and the size of the electrophoresis apparatus can be reduced, the electrophoresis time can be shortened, and high throughput can be achieved. is there.

  In the first invention, since the pH gradient with respect to the migration direction of the gel for isoelectric focusing satisfies the relationship of “a <b” and “b> c” as described above, the above effect can be obtained. .

  In the second invention, more specifically, 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, and c is 0.00. It is limited to the range of 15 to 0.3, and the effect of the first invention can be realized more preferably.

  According to the third invention, the isoelectric focusing method can be performed with the effects of the first invention and the second invention.

(Effect of the fourth invention)
By performing the isoelectric focusing method of the third invention as the first-dimensional electrophoresis in the two-dimensional electrophoresis, in addition to ensuring the effect of the third invention, the second-dimensional SDS-PAGE, etc. Since the width of the separation gel to be used can be reasonably shortened, the two-dimensional electrophoresis apparatus can be downsized without impairing the high resolution.

(Effect of the fifth invention)
As specified in the fifth invention, the specimen used for the isoelectric focusing method according to the third or fourth invention is preferably a biological cell extract for the reason described above with respect to the first invention. Animal cell extracts are particularly preferable, and human cell extracts are particularly preferable.

The result of the two-dimensional electrophoresis which concerns on 1st Example is shown.

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

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

[Isoelectric focusing gel]
The gel for isoelectric focusing of the present invention has a gel length in the range of 5 to 10 cm, more preferably in the range of 5 to 8 cm. The pH range of the gel ranges from 3-10. Furthermore, the pH gradient of the gel with respect to the migration direction is such that “a <b” and “b> c” when the gel length up to pH 5 is a, the gel length at pH 5-7 is b, and the gel length above pH 7 is c. 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, and c is It is within the range of 0.15 to 0.3, and more preferably satisfies the relationship of “a + c ≦ b”. Such setting of the pH gradient of the gel corresponds to the distribution of isoelectric points of various proteins contained in the aforementioned extract of biological cells.

  In the isoelectric focusing method of the present invention, the type of gel used for electrophoresis is not particularly limited. For example, amphoteric carrier (carrier ampholite) is added to polyacrylamide gel, and an electric field is applied to form a desired pH gradient, or monomer derivatives such as acrylamide derivatives having various isoelectric side chains are used. A gel prepared by a method (IPG method) for forming a pH gradient in a fixed manner simultaneously with the preparation of a gel such as a polyacrylamide gel is preferably used. The type of gel is not limited as long as it can be used as an isoelectric focusing gel, and for example, polyacrylamide gel, agarose gel and the like can be preferably exemplified.

  Protein synthesis takes place intracellularly. In general, since various biochemical environments such as pH are different between the inside and outside of a cell, the properties of a protein remaining in the cell and a protein secreted outside the cell are different. The cause is presumed to be an amino acid sequence or post-translational modification. Thus, by paying attention to the characteristics of the protein in the specimen, the pH gradient of the gel for isoelectric focusing can be changed in a purposeful manner.

[Isoelectric focusing method]
In the isoelectric focusing method of the present invention, the instrument used for 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 isoelectric focusing is not particularly limited, but it is necessary to pay attention to the electrophoresis protocol in order to achieve high resolution and high throughput. In the stage of preparing the specimen solution, it is preferable to remove as much as possible the coarse substances which are charged substances that are not subject to separation / purification. 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, by performing a constant voltage process in which a relatively weak voltage is applied for about one and a half hours to three and a half hours at an early stage of the isoelectric focusing protocol, it is possible to remove coarse substances without applying a load to the apparatus. . If a high voltage is used in this step, the coarse substance moves rapidly to the electrode side, and a strong current flows, so the load is applied to the instrument and the separation of each protein becomes worse (the spots in the gel There is a risk of clogging).

  In isoelectric focusing, a constant voltage step was performed by applying a constant voltage with a value in the range of 100 V to 600 V per gel containing the specimen, and the current change width per 30 minutes of electrophoresis was within the range of 5 μA. It is preferable that a voltage raising step for raising the voltage from the constant voltage later is started, and the final voltage in the voltage raising step is within a range of 3000V to 6000V. Further, it is preferable to keep the gel temperature constant 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, the positively charged rough matter is quickly moved to the cathode, and the negatively charged rough matter is quickly moved to the anode by performing the constant voltage process at a low constant voltage of 100V to 600V before the voltage starts to rise. . 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.
[Two-dimensional electrophoresis]
The isoelectric focusing method of the present invention can also be carried out as first-dimensional electrophoresis in two-dimensional electrophoresis. In this case, the second-dimensional electrophoresis is not necessarily limited, but SDS-PAGE is preferable. If the first-dimension isoelectric focusing is performed with a small device, and has high resolution and high throughput, the second-dimension electrophoresis can also reduce the size of the device and achieve high resolution and high throughput. realizable. Therefore, the present invention can be applied not only to isoelectric focusing performed independently but also to the first-dimensional electrophoresis in two-dimensional electrophoresis. When performing two-dimensional electrophoresis, SDS-PAGE is preferably performed following isoelectric focusing, so the second-dimensional SDS-PAGE will be described below.
[Second-dimensional SDS-PAGE]
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 PAG (polyacrylamide gel) for performing SDS-PAGE, the total concentration (T%) of acrylamide as a monomer and a crosslinking agent, the ratio of the crosslinking agent in the total weight of acrylamide and the crosslinking agent (C%), etc. Is not particularly limited.
[Gel concentration at the base end of the gel for the second dimensional electrophoresis]
When the gel length of the gel for the first dimension electrophoresis is set short, SDS-PAGE performed as the second dimension has a gel concentration of about 3 to 6% at the proximal end in the electrophoresis direction in the electrophoresis gel. It is preferable that the concentration is low. The gel concentration directly means the monomer concentration at the time of the polymerization reaction of the gel. However, the higher the monomer concentration at the time of the polymerization reaction, the denser the network structure of the gel. Means density.

  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 second-dimensional electrophoresis gel is high (the gel network is dense), the protein concentrated in the spot is transferred to the second-dimensional electrophoresis gel. High barrier property against migration, protein migration leakage becomes remarkable, and spots broaden laterally with respect to the migration direction. Such a problem is solved by the above embodiment.

  In SDS-PAGE, SDS (sodium dodecyl sulfate), which is a surfactant, is added to a sample to unravel the higher-order structure of the protein contained in the sample, and the charge of amino acid residues of the protein is also relatively reduced by SDS. Thus, electrophoresis is performed using the molecular sieving effect.

[Sample preparation]
The specimen applied to the isoelectric focusing 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.

In the isoelectric focusing or two-dimensional electrophoresis of the third invention or the fourth invention described above, it is preferable that the specimen is an extract of a biological cell, particularly an animal cell, particularly a human cell. In isoelectric focusing, 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.

  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 touch the sieve, so it 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. Therefore, in the preparation of the specimen, it is preferable to remove coarse substances that are not subject to separation / purification in order to reduce the load on the instrument 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.

  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 (surfactant (Octylphenoxy) polyethoxyethanol manufactured by SIGMA-ALDRICH)
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 was dissolved in 130 μl of DeStreak Rehydration Solution (GE), which is a swelling buffer for the gel for first-dimensional isoelectric focusing, and used for first-dimensional isoelectric focusing. A sample solution (a sample 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 is a rod-like gel having a length of 7 cm and a diameter of about 0.3 cm, T = 4%, C = 3%, and has the following pH gradient characteristics.
pH range: 3-10
Gel length at pH 3-5: 1.7 cm
Gel length at pH 5-7: 3.6 cm
Gel length at pH 7-10: 1.7 cm
(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 from both ends of the gel. It is. Silicon oil poured from both ends spread toward the inside of the gel. With the silicone oil covering the gel, the specimen solution was allowed to penetrate the gel overnight at room temperature. After that, 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 750 Vhr at a constant voltage of 300 V (current for 30 minutes before the end of the step (2) The voltage was gradually increased to 1000V over 300Vhr), (3) the voltage was gradually increased to 5000V over 4500Vhr, and (4) the total Vhr at 5000V constant voltage thereafter. The first-dimension isoelectric focusing was performed until the value reached 12000.

(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 dimension electrophoresis, NuPAGE 4-12% Bis-Tris Gels manufactured by Invitrogen 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.) is used in the electrophoresis buffer. An agarose solution for adhesion in which an appropriate amount of BPB (bromophenol blue) was dissolved was used.

  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 98% (v / v) 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. In the upper end of FIG. 1, the pH gradient of the isoelectric focusing gel used in the first dimension is shown by a scale, and the molecular weight (KDa) of the marker is shown in the left end of FIG.

[Second Embodiment]
In the second example, 2D-DIGE was performed. In the second embodiment, among the procedures described in the first embodiment, the procedure in the section “(Preparation of specimen solution)” is changed to the procedure in the section “(Preparation of specimen solution in 2D-DIGE)” below. 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 an additional 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 to the first embodiment]
A 7-cm long 1-dimensional isoelectric focusing gel was prepared in the same manner as in the section “(Preparation of 1-dimensional isoelectric focusing gel)” in the first example. However, this gel differs from the first embodiment in that the pH gradient is almost linear as a whole as described below.
pH range: 3-10
Gel length at pH 3-5: 2.1 cm
Gel length at pH 5-7: 2.45 cm
Gel length at pH 7-11: 2.45 cm
In this comparative example, all steps from the preparation of the specimen to the fluorescent staining and analysis of the gel are the same as in the first embodiment, except that the gel is used as the first-dimensional isoelectric focusing gel. went.

  The results of two-dimensional electrophoresis in this comparative example are shown in FIG. At the upper end of FIG. 2, the pH gradient of the isoelectric focusing gel used in the first dimension is shown by a scale, and the molecular weight (KDa) of the marker is shown at the left end of FIG.

When FIG. 2 is compared with FIG. 1, the following points can be recognized.
(1) The clogging of spots in the pH 5 to 7 region (acid to neutral region) seen in FIG. 2 as a whole is well separated in FIG. For example, in the region where the molecular weight is around 70 KDa and the pH is around 5 to 5.5, fine spot separation is seen in FIG. 1, but spot separation is insufficient in FIG. The same indication can be made in the region of a molecular weight of around 45 KDa and a pH of around 5.5.
(2) As a whole, in FIG. 2, excessive separation ability is recognized in the pH 7 to 10 region (neutral to basic region), and in this pH region, the separation ability of the degree shown in FIG. is there. Specifically, for example, this is based on the comparison of the spot separation ability of FIG. 1 and FIG. I can confirm.

The present invention provides an isoelectric focusing gel in which the gel length is shortened without impairing high resolution, and an isoelectric focusing method using the same.

Claims (4)

First-dimension isoelectric focusing that satisfies the following condition (1) is performed, equilibration buffer treatment that satisfies the following condition (2) is performed, and the second-dimensional SDS- that satisfies the following condition (3): A two-dimensional electrophoresis method characterized by performing PAGE.
(1) The specimen is an extract of human keratinocytes,
The gel length is in the range of 5 to 10 cm, the pH range of the gel is 3 to 10, the pH gradient of the gel with respect to the migration direction is a, the gel length up to pH 5 is b, the gel length of pH 5-7 is b, When the gel length of pH 7 or higher is c and 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, and c is 0.15 to 0.15. This is carried out using an isoelectric focusing gel within the range of 0.3.
(2) The gel subjected to the isoelectric focusing is treated with an equilibration buffer containing a reducing agent, and then treated with an equilibration buffer containing an alkylating agent.
(3) Perform using Bis-Tris Gel. The two-dimensional electrophoresis method according to claim 1, wherein the reducing agent is DTT and the alkylating agent is iodoacetamide. 3. The two-dimensional electrophoresis method according to claim 1, wherein a gel concentration at a base end portion in the migration direction of the Bis-Tris Gel is 3 to 6%. The two-dimensional electrophoresis method according to claim 1 or 2 , wherein the gel concentration of the Bis-Tris Gel is 4 to 12%.