WO2016002282A1

WO2016002282A1 - Gel buffer solution for electrophoresis and polyacrylamide gel for electrophoresis

Info

Publication number: WO2016002282A1
Application number: PCT/JP2015/060426
Authority: WO
Inventors: 弘子藤生; 篤浅川; 入江　勉
Original assignee: アトー株式会社
Priority date: 2014-07-04
Filing date: 2015-04-02
Publication date: 2016-01-07
Also published as: JP6453326B2; JPWO2016002282A1

Abstract

This gel buffer solution for electrophoresis contains: a buffering agent selected from among tris(hydroxymethyl)- aminomethane, bis(2-hydroxyethyl)iminotris(hydroxymethyl)- methane, monoethanolamine, diethanolamine and triethanolamine; glycine and at least one zwitter ion having an acid dissociation constant (pKa) satisfying 9.6 ≤ pKa ≤ 11; and at least one acid. This gel buffer solution for electrophoresis has a pH within the range from 5.5 to 7.5.

Description

Gel buffer for electrophoresis and polyacrylamide gel for electrophoresis

(Cross-reference of related fields)
This application claims the benefit of priority of Japanese Patent Application No. 2014-139196 filed on Jul. 4, 2014, the entirety of which is incorporated herein by reference.
(Technical field)
The present invention relates to a gel buffer for electrophoresis and a polyacrylamide gel for electrophoresis.

Separation of molecules such as polypeptides, proteins or nucleic acids by polyacrylamide gel electrophoresis is a fundamental system that separates the molecules according to their molecular weight, and is a fundamental technology indispensable for life science research. is there. The electrophoretic system was invented by Ornstein (Non-patent Document 1) and Davis (Non-patent Document 2) in the 1960s, and later improved by Laemmli (Non-patent Document 3). The Laemmli method is currently the most used SDS (dodecyl sulfate). Sodium) -polyacrylamide gel electrophoresis system. However, the Laemmli method gel consists of a basic gel buffer solution of 375 mM Tris / pH 8.8, and the main component of the gel, acrylamide, is unstable and hydrolyzed under basic conditions. Was not suitable.

Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) gel recently invented by Updyke et al. Is a neutral gel buffer, a neutral electrophoresis buffer (Tris-MOPS). Since the system is combined with a buffer solution or a Tris-MES buffer solution, gels can be stored for a long period of time, and the environment during electrophoresis can be kept neutral (Patent Document 1). On the other hand, an electrophoretic gel has been developed that can be stored for a long time just by neutralizing the pH of the gel buffer (Tris) of the Laemmli method (Patent Document 2), but Tris-HEPES is used as the electrophoresis buffer. It was necessary to use a buffer. In this way, the molecules cannot be separated using the Laemmli electrophoresis buffer that has been used in any of the systems, and the reduced protein is not reoxidized because the environment during the migration is neutral. Thus, it is necessary to add a reducing agent. In addition, protein mobility and fractional molecular weight ranges do not match those of the Laemmli system, and thus cannot be easily correlated with data accumulated so far.

Therefore, as a weakly acidic gel that can use Laemmli's electrophoresis buffer, electrophoresis includes an ampholyte composed of Tris, glycine, and a coexisting ampholyte having a base dissociation constant pKb of 8.3 <pKb <9.6. A polyacrylamide precast gel for use is disclosed in Patent Document 3, and this precast gel has no change in gel shape and electrophoretic image from half a year to one year or more. However, a technique relating to high-speed electrophoretic separation under high voltage and / or high current conditions is not disclosed.

US Pat. No. 5,578,180 U.S. Pat. No. 6,733,647 Patent No. 394001

Actually, when the gel described in Patent Document 3 was used for high-speed electrophoresis under high voltage conditions, it was found that an interface was formed on the gel and the shape of the band was disturbed. High-speed electrophoretic separation technology under high voltage and / or high current conditions will never get high resolution while maintaining band shape and mobility because gels and running buffers are exposed to high temperatures during electrophoresis. It's not easy.

Therefore, in the present invention, the quality can be stably maintained over a long period of time of refrigerated for more than half a year, and various electrophoresis buffers including Laemmli electrophoresis buffer and other buffers (Tris-MOPS etc.) are used for electrophoresis. A gel buffer solution that can be obtained and that provides high resolution without causing an interface on the gel under high voltage and / or high current electrophoresis conditions, and an electrophoresis gel prepared using the gel buffer provide.

The present inventors have intensively studied to solve the above problems, and using glycine as an amphoteric electrolyte for electrophoresis gel buffer, combined with it, the acid dissociation constant pKa is 9.6 ≦ pKa ≦ 11. It has been found that when different types of zwitterions are used, high resolution can be obtained without causing an interface on the gel even when high-speed electrophoresis is performed under high voltage and / or high current conditions.
In addition, the numerical value of the acid dissociation constant pKa of the zwitterion in this specification is determined based on the description in CRC handbook of Chemistry and Physics, 90 ^th edition, internet version 2010.
The present invention is as follows.

[1] A gel buffer for electrophoresis, wherein the buffer is selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine A gel buffer for electrophoresis having a pH of 5.5 to 7; and an agent; glycine and at least one zwitterion having an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11; and at least one acid A gel buffer for electrophoresis that is between .5.

[2] The electrophoresis according to Item 1, wherein the concentration of the buffer is 50 mM to 300 mM, the concentration of the glycine is 10 mM to 1000 mM, and the concentration of the at least one zwitterion is 10 mM to 200 mM. Gel buffer.

[3] Item 1 or 2 wherein the zwitterion is selected from alanine, arginine, cysteine, aspartic acid, glutamic acid, proline, γ-carboxyglutamic acid, γ-aminobutyric acid, 2-aminoisobutyric acid, 6-aminocaproic acid, and CAPS. 2. A gel buffer for electrophoresis as described in 2.

[4] The gel buffer for electrophoresis according to any one of items 1 to 3, further comprising one or more additives selected from sugars, polyhydric alcohols, and salts.

[5] A polyacrylamide gel for electrophoresis produced using the gel buffer solution for electrophoresis according to any one of items 1 to 4.

[6] A buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine; glycine and an acid dissociation constant pKa of 9 A polyacrylamide gel for electrophoresis, comprising: at least one zwitterion satisfying 6 ≦ pKa ≦ 11; and at least one acid.

[7] The polyacrylamide gel for electrophoresis according to item 5 or 6, which is a precast gel.

[8] A method for separating a polypeptide, protein or nucleic acid using the polyacrylamide gel for electrophoresis according to any one of items 5 to 7.

[9] Acrylamide; Crosslinker; Buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine; glycine and acid Polymerization of a mixture containing at least one zwitterion with a dissociation constant pKa of 9.6 ≦ pKa ≦ 11; and at least one acid under the condition that the pH is in the range of 5.5 to 7.5 A method for producing a polyacrylamide gel for electrophoresis, which comprises polymerizing in the presence of an agent.

Since the gel buffer of the present invention and the gel for electrophoresis prepared using the gel buffer are stable for a long period of time, a large amount of precast gel that maintains uniform and long-term stable quality is produced and stored. I can do it. Therefore, it is possible to supply the precast gel quickly when necessary.

In addition, if the gel buffer of the present invention is used, the conventionally used Laemmli method electrophoresis buffer can be used, so data accumulated so far in the Laemmli method (similar fractional molecular weight range, molecular mobility). Therefore, the reliability of the data is improved. In addition, since an electrophoresis buffer other than the Laemmli method can be used, it is excellent in versatility.

Furthermore, the polyacrylamide gel prepared using the gel buffer of the present invention does not disturb the shape and / or pattern of the electrophoresis band even under high voltage and / or high current conditions, and the interface on the gel is not affected. High resolution can be obtained without causing it.

2 shows electrophoresis patterns of zwitterion-containing gels having different acid dissociation constants. A: Gel conforming to Laemmli method, B: Zwitterion-free gel, C: Serine-containing gel, D: Taurine-containing gel, E: Alanine-containing gel, F: Proline-containing gel, G: 6-aminocaproic acid-containing gel . c: chicken muscle extract, p: human plasma sample, m: molecular weight marker. Arrow: indicates an interface. Figure 5 shows the interface on the gel resulting from electrophoresis using zwitterion-containing gels with pKa <9.6. C ′: serine-containing gel, D ′: taurine-containing gel, E ′: alanine-containing gel. Square (broken line): C ′ and D ′ indicate interfaces. E ′ has no interface. Electrophoresis patterns migrated under constant voltage conditions of 150V, 300V, and 500V are shown. A is energized at 150V for 80 minutes, B is energized at 300V for 30 minutes, and C is energized at 500V for 15 minutes. c: chicken muscle extract, m: molecular weight marker, p: human plasma sample.

Hereinafter, preferred embodiments of the composition and method of use of the gel buffer for electrophoresis of the present invention and the composition and method of use of the polyacrylamide gel for electrophoresis produced using the gel buffer for electrophoresis will be described. .

The gel buffer for electrophoresis of the present invention is a buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine; Glycine and at least one zwitterion with an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11; and at least one acid; and the pH of the gel buffer for electrophoresis is 5.5 to 7.5 Between.

As the buffer, tris (hydroxymethyl) aminomethane is preferable. The concentration of tris (hydroxymethyl) aminomethane may be usually 50 mM to 300 mM, preferably 70 mM to 150 mM, more preferably 80 mM to 100 mM in the gel buffer. If the Tris concentration contained in the gel buffer is high, the low molecular region cannot be detected, and the current increases, so that the gel undergoing electrophoresis becomes hot and the band shape is disturbed.

Glycine and at least one zwitterion having an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11 are both amphoteric electrolytes. The ampholyte has an acidic functional group and a basic functional group in the molecule, and at pH = pKa value, the functional group dissociates and the positive and negative charge amounts become equal to become electrically neutral. . At pH lower than the pKa value, the acidic functional group is positively charged and becomes a cation, and at pH higher than the pKa value, the basic functional group is negatively charged and behaves as an anion. After the start of energization, the acid contained in the electrophoresis gel is ionized and moves to the anode side, and the pH of the gel increases when the gel escapes from the electrophoresis buffer. Then, the ampholyte starts to move to the anode because it is negatively charged at a pH higher than its own pKa value. The amphoteric electrolyte with a lower pKa value starts moving to the anode side earlier. Since the pKa value of glycine contained in the gel of the present invention is 9.6, if the pKa value of the coexisting zwitterion is larger than 9.6, it will move to the anode side later than glycine. Thus, it is considered that electrophoresis without causing an interface is possible without the potential gradient in the gel reaching equilibrium.

The concentration of glycine is usually 10 mM to 1000 mM in the gel buffer, preferably 100 mM to 500 mM, more preferably 180 mM to 300 mM. When the concentration of glycine contained in the gel buffer is extremely low, the fractionation range is narrowed and the separation of the high molecular weight region is improved, but the separation of the low molecular weight region is deteriorated. The concentration of glycine occupies 10% or more, preferably 50% or more, more preferably 70% or more, and particularly preferably 80% or more of the ampholyte in the gel buffer in molar ratio.

The at least one zwitterion may be a compound having an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11 or a derivative thereof, specifically, alanine, arginine, cysteine, aspartic acid, glutamic acid, proline, Examples include amino acids such as γ-carboxyglutamic acid, γ-aminobutyric acid, 2-aminoisobutyric acid, 6-aminocaproic acid, CAPS (3- (cyclohexylamino) -1-propanesulfonic acid), and combinations thereof. It is not limited. The concentration of the at least one zwitterion is usually between 10 mM and 200 mM, preferably between 20 mM and 100 mM, more preferably between 20 mM and 50 mM in the gel buffer. According to the present invention, when the acid dissociation constant pKa of zwitterion is 9.6 ≦ pKa ≦ 11, the amphoteric electrolyte is formed on the gel in the separation by high-speed electrophoresis under high voltage and / or high current conditions. High resolution can be obtained without causing the resulting interface. The acid dissociation constant pKa of the zwitterion is preferably 9.6 <pKa <11.

At least one acid contained in the gel buffer of the present invention keeps the pH of the gel buffer from weakly acidic to a neutral region and at the same time sharpens the shape of the band and improves the control of the electrophoresis speed and the separation of the band. There is work. Acids include, but are not limited to, weak acids such as citric acid, glycolic acid, maleic acid, phosphoric acid, acetic acid and boric acid, and strong acids such as hydrochloric acid, sulfuric acid and nitric acid. A combination of one or two or more weak acids and weak acids, weak acids and strong acids, or strong acids and strong acids may be used. In particular, acetic acid, sulfuric acid, and hydrochloric acid are preferable, and a combination of acetic acid and hydrochloric acid is most preferable. Further, at least one acid may be added as a salt of the buffer. Since the acid is used to adjust the pH of the gel buffer, the concentration is not particularly limited, but is usually 10 mM to 300 mM in the gel buffer. The coexisting concentrations are preferably in the range of 10 mM to 50 mM acetic acid and 10 mM to 100 mM hydrochloric acid, for example.

The pH of the gel buffer solution neutralized with acid may be in the range of 5.5 to 7.5, preferably pH 5.5 to 7.0, more preferably pH 6.0 to 6.5. Fit in. pH 7. If it is higher than 5, hydrolysis of the polyacrylamide gel tends to proceed, and the shelf life cannot be extended. When the pH is lower than 5.5, the hydrolysis of the polyacrylamide gel hardly proceeds, but the molecular migration image becomes unclear. By adjusting the pH to 5.5 to 7.5, the hydrolysis rate of acrylamide is decreased, and the performance and shape of the gel are stable for a long period of time of refrigeration for more than half a year, particularly for more than one year.

The content ratio of the buffering agent and at least one acid is preferably 15: 1 to 1: 3 by weight. The total content of glycine and at least one zwitterion satisfying 9.6 ≦ pKa ≦ 11 and the content ratio of at least one acid are preferably 25: 1 to 2.5: 1 by weight.

The gel buffer of the present invention includes a sugar (eg, sucrose, glucose, galactose, fructose, sorbitol, trehalose, dextran), a polyhydric alcohol (eg, glycerol, ethylene glycol, propylene glycol), a salt containing an organic salt and an inorganic salt. (E.g., sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, and potassium phosphate, sodium citrate, sodium acetate, magnesium sulfate, calcium chloride, ammonium chloride, ammonium sulfate), etc. May be included.

The present invention also includes a polyacrylamide gel for electrophoresis prepared using the gel buffer for electrophoresis of the present invention described above. Polyacrylamide gels for electrophoresis include precast gels that are provided to users in the form of pre-formed gels.

Such polyacrylamide gel for electrophoresis contains the buffer, glycine, at least one zwitterion having an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11, and at least one acid.

The method for producing the polyacrylamide gel for electrophoresis of the present invention can be produced according to a conventionally known method for producing polyacrylamide gel for electrophoresis using the gel buffer of the present invention.

In one embodiment, the polyacrylamide gel for electrophoresis comprises acrylamide, a crosslinking agent, a buffer, glycine and at least one zwitterion with an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11, and at least one Is prepared by polymerizing in the presence of a polymerization initiator under conditions where the pH is in the range of 5.5 to 7.5.

The crosslinking agent is added for the purpose of crosslinking acrylamide. For example, N, N '-methylenebisacrylamide (BIS), N, N'-allyltartaric acid amide (DATD), dihydroxyethylene-bisacrylamide (DHEBA). Water-soluble divinyl compounds such as) can be used.

Buffer, glycine and at least one zwitterion, and at least one acid are as described above for the gel buffer for electrophoresis.

Polymerization initiators that are catalysts for polymerizing gels include oxidizing agents such as ammonium persulfate (APS) and potassium persulfate (KPS) and N, N, N ′, N′-tetramethylethylenediamine (TEMED) and the like. Although a reducing agent can be used together, it is not limited to these. Further, photopolymerization using riboflavin or the like may be performed together with polymerization by a polymerization initiator. The oxidizing agent and reducing agent are usually used in an amount of 0.05% to 5% (weight / volume) based on the total amount of monomers to be polymerized.

The above mixture may contain a water-soluble polymer which is a carrier added for the purpose of giving elasticity and strength to the gel. Examples of such a water-soluble polymer include agarose, polyvinyl alcohol, polyethylene glycol, Polyvinyl pyrrolidone, polymethyl vinyl ether and the like are included.

The type of support for producing the gel is not particularly limited. Examples include glass, plastic, and ceramic. Various types of gel preparation plates are commercially available as supports for preparing gels, and there are a variety of materials and chemical modification of the surface. The composition of the optimal gel solution often varies depending on the type of gel preparation plate, but the gel buffer of the present invention can be used in any gel preparation plate and is highly versatile.

The electrophoresis buffer is composed of a combination of an amine buffer and an ampholyte, and may contain a surfactant. In addition to Laemmli's tris-glycine-sodium dodecyl sulfate (SDS) buffer, tris, triethanolamine, tricine, 3-morpholinopropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), N- ( 2-acetamido) -2-aminoethanesulfonic acid (ACES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), acetic acid, boric acid And buffers containing these and combinations thereof, and interfaces therewith Such as sexual agent further the added buffer is not limited to be used. As the amphoteric electrolyte constituting the electrophoresis buffer, known amphoteric electrolytes such as glycine and the above zwitterions can be used.

The present invention also includes a method for separating a polypeptide, protein or nucleic acid using the polyacrylamide gel for electrophoresis of the present invention described above. In the present specification, a combination of 2 or more and 50 or less amino acids is referred to as a polypeptide, and a combination of 51 or more amino acids is referred to as a protein. Nucleic acids include DNA and RNA.

The polyacrylamide gel for electrophoresis of the present invention can be stored for a long period of time, and is suitable for separation of polypeptides, proteins or nucleic acids. In addition, in electrophoresis performed under high voltage and / or high current conditions, molecules move at a higher speed than in the past, but even in this case, the shape and / or pattern of the electrophoresis band is not disturbed, and the interface is formed on the gel. High resolution can be obtained without causing it. The “high voltage condition” means that the electrophoresis voltage is 20 V / cm or more per unit length between the electrodes of the gel. Although the upper limit of a voltage is not specifically limited, Usually, it is 80 V / cm or less. “High current condition” means that the electrophoretic current is 40 mA / cm ² or more per unit cross-sectional area of the current-carrying surface of the gel. The upper limit of the current is not particularly limited, but is usually 250 mA / cm ² or less.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1
Acid dissociation constant In order to investigate the effect of pKa on the mobility and electrophoresis pattern of proteins, an electrophoresis gel containing zwitterions with a pKa of 9 to 11 was prepared and electrophoresed. Compared with.

The separation gel is the gel buffer shown in Table 1, acrylamide and bis-acrylamide (10% T / 3% C, T = acrylamide and the total mass of N, N′-methylenebisacrylamide (BIS), C = acrylamide. And the ratio of BIS to the total of BIS, the same applies hereinafter), and polymerization was started by adding 0.08% APS and 0.03% TEMED. The concentrated gel consists of the gel buffer shown in Table 1, acrylamide and bis-acrylamide (4.5% T / 3% C), and was prepared by polymerization by adding 0.08% APS and 0.03% TEMED. . As the electrophoresis buffer, 25 mM Tris, 192 mM glycine, and 0.1% SDS (based on the Laemmli method) were used. Electrophoresis was performed using an electrophoresis apparatus (AE6530) manufactured by Atto Co., Ltd., and energized for 30 to 35 minutes at a constant voltage of 300 V (the electrophoresis voltage was 34 V / per unit length between the gel electrodes). cm, the electrophoretic current corresponds to 40 to 75 mA / cm ² per unit cross-sectional area of the current-carrying surface of the gel).

The protein sample was treated with EzLabel FluoroNeo (Ato Inc., WSE-7010) to produce fluorescently labeled protein and unlabeled protein. The gel after electrophoresis was stained with EzStain AQUA (Ato, AE-1340) after obtaining a fluorescent image excited with a blue LED. The dyed gel was analyzed with CS Analyzer (Atto Corporation) after capturing an image with a scanner. The relative mobility Rf (%) was calculated by (distance from top of separation gel to each band position) / (distance from top of separation gel to electrophoresis tip) × 100.

The electrophoretic results are shown in FIG. 1, and the relative mobility (%) of the molecular weight marker protein is shown in Table 2. 1A to D are Laemmli gels (A) as controls, trisglycine gels (B) not containing zwitterions, gels (C) containing serine (pKa: 9.15) having a pKa of less than 9.6, And the result of the taurine (pKa: 9.06) containing gel (D) is shown. (E) to (G) are zwitterion-containing gels having a pKa of 9.6 to 11, and (E) is alanine (pKa: 9.6) and (F) is proline (pKa: 10.6), respectively. , (G) is a result of electrophoresis of a gel containing 6-aminocaproic acid (pKa: 10.8) at a concentration of 30 mM.

Samples were chicken muscle extract (c), human plasma (p), and molecular weight marker (m) (chicken muscle-derived myosin: 220 kDa, E. coli-derived β-galactosidase: 116 kDa, rabbit muscle-derived phosphorylase B: 97 kDa, derived from bovine serum. Albumin: 66 kDa, chicken egg-derived ovalbumin: 45 kDa, bovine blood cell-derived carbonyl anhydrase: 30 kDa, soybean-derived trypsin inhibitor: 20 kDa, bovine milk-derived α-lactalbumin: 14.4 kDa).

In the gel (B) containing no zwitterion, the low molecular region could not be separated. Serine (pKa: 9.15) -containing gel (C) and taurine (pKa: 9.06) -containing gel (D) showed the same electrophoretic pattern as the Laemmli method gel, but around 45 kDa on the gel. An interface was formed (arrow part in FIG. 1, C ′ and D ′ in FIG. 2), and the protein before and after that could not be separated, and it was difficult to confirm the separated band.

Gels (E)-(G) containing zwitterions with a pKa of 9.6 to 11 show an electrophoresis pattern equivalent to that of Laemmli method gels, and have the same molecular weight range and protein mobility. In addition, an interface caused by zwitterions did not occur on the gel (E ′ in FIG. 2), and separation by electrophoresis at a high voltage was good.

Example 2
In order to verify the change in protein mobility depending on the concentration of alanine, which is one of zwitterions, a gel was prepared using the gel buffer described in Table 3, and the same as in Example 1 was performed. Electrophoresis was performed by the method. As shown in the relative mobility (%) of molecular weight markers in Table 4 and the electrophoresis pattern (photograph is not shown), the electrophoretic pattern is not affected even when the alanine concentration is changed from 25 mM to 100 mM. The Laemmli method The same molecular weight range and protein mobility as those of the gel were observed, and good separation was observed.

Example 3
In order to verify the change in protein mobility dependent on the Tris concentration, a gel was prepared using the gel buffers described in Table 5 and Table 7, and electrophoresis was performed in the same manner as in Example 1. It was. Table 6 shows the results when the Tris concentration was changed from 50 mM to 80 mM, and Table 8 shows the results when the Tris concentration was 80 mM or more. As shown in the relative mobility and electrophoresis pattern (photos not shown) in Tables 6 and 8, good separation was observed in both cases. However, when the Tris concentration was 70 mM or less, the molecular weight on the low molecule side was observed. Since the range of the image was expanded, the 45 kDa band of the molecular weight marker was divided into two (A to C). Since the 45 kDa band is phosphorylated protein with ovalbumin, if the separation on the low molecular side is improved with a high concentration acrylamide gel or the like, the phosphorylated and dephosphorylated band may be separated. Tris with a concentration of 80 mM or more showed a fractional molecular weight range and protein mobility equivalent to the Laemmli gel (E to H), but gradually increased separation on the polymer side as the Tris concentration increased (G, H). Thus, there was a tendency for the fractional region on the low molecular side of 30 kDa or less to be narrowed. On the other hand, addition of sulfuric acid as an acid other than acetic acid (E to H) did not significantly affect the electrophoresis pattern.

Example 4
In order to verify the change in protein mobility dependent on glycine concentration, a gel was prepared using the gel buffer described in Table 9, and electrophoresis was performed in the same manner as in Example 1. As shown in the relative mobility of Table 10 and the electrophoresis pattern of FIG. 5 (the gel photo is not shown), the separation of the polymer region is better when the glycine concentration is high, and low when the glycine concentration is low. It was found that the separation of the molecular side region was improved. In this way, the protein mobility changes depending on the glycine concentration.By selecting the glycine concentration that matches the target molecular region, it is possible to separate and detect the band of the target molecular region in more detail. is there. In addition, in the gel (CF) containing glycine at a concentration of 100 mM or more, the electrophoresis pattern and relative mobility of the protein were the same as those of the Laemmli method gel, and good separation was observed.

Example 5
In order to verify the change in protein mobility depending on the acetic acid concentration, a gel was prepared using the gel buffer described in Table 11, and electrophoresis was performed in the same manner as in Example 1. As a result, as shown in Table 12 relative mobility and electrophoresis pattern (gel photo is not shown), the protein migration pattern and relative mobility are the same as the Laemmli gel, and good separation is observed. It was done. In addition, it was shown that as the acetic acid concentration was increased, the separation on the polymer side was slightly improved, the band became sharper, and the migration speed gradually decreased.

Example 6
In order to verify the effect of adding additives to a gel consisting of tris, glycine, zwitterion, acid, triethanolamine (TEA, pKa: 7.76), γ- A gel was prepared using a gel buffer to which aminobutyric acid (GABA, pKa: 10.43) was added, and electrophoresis was performed in the same manner as in Example 1. As a result, as shown in Table 14 and the electrophoresis pattern (gel photograph not shown), good separation was observed in both cases. The addition of GABA had almost no effect on the electrophoretic pattern and mobility (C), but the addition of TEA showed that the fractional area on the low molecular side was narrowed slightly (B, D). ).

Example 7
Further, in order to verify the influence of sucrose or glycerol often used as an additive in the gel, a gel was prepared using the gel buffer described in Table 15, and electricity was obtained in the same manner as in Example 1. Electrophoresis was performed. As shown in the relative mobility and electrophoretic pattern in Table 16 (the gel photo is not shown), all showed good separation, almost no effect from the addition of glycerol and sucrose, equivalent to the Laemmli gel. The molecular weight range and protein mobility were shown.

Example 8
In order to verify long-term storage stability, heat treatment was performed at 4 ° C., 37 ° C., and 50 ° C., and an accelerated stability test of the gel was performed. It has been shown that a one day warming treatment at 37 ° C. is equivalent to a one month warming treatment at 4 ° C., which is a typical storage temperature. 10% T3% C acrylamide and 10% T3.3% C acrylamide gels were prepared using the gel buffer described in Table 17 and heated at 4 ° C., 37 ° C., 50 ° C. for 1 week. (Table 18), electrophoresis was performed in the same manner as in Example 1. As shown in the relative mobility of the molecular weight marker (Table 19) and the electrophoresis pattern (the gel photo is not shown), the relative mobility of each protein is hardly changed, and the electrophoresis pattern is also highly resolvable, No significant difference due to the heating treatment was observed.

Example 9
In the same manner as in Example 8, in order to verify long-term storage stability, the gels were heated at 4 ° C., 37 ° C., and 50 ° C., and an accelerated stability test was performed on the gel. Equivalent to one month at ℃). A 10% T3.3% C acrylamide gel was prepared using the gel buffer described in Table 20 and heated at 4 ° C., 37 ° C., 50 ° C. for 9 days and 14 days (Table 21). ) And electrophoresis was performed in the same manner as in Example 1. As shown in the relative mobility of the molecular weight marker (Table 22) and the electrophoresis pattern (the gel photo is not shown), the relative mobility of each protein is hardly changed, and the electrophoresis pattern is also highly resolvable, No significant difference due to the heating treatment was observed.

Example 10
In order to investigate the influence of the voltage when performing electrophoresis, a gel was prepared with the composition shown in Table 23, and electrophoresis was performed in the same manner as described above under constant voltage conditions of 150 V, 300 V, and 500 V. As shown in FIG. 3, there is no difference in the patterns electrophoresed for 80 minutes at a commonly used voltage of 150 V and for 30 minutes at a high voltage of 300 V, and the band shape and mobility are the same. It was done. When electrophoresis was performed at a higher voltage of 500 V, the mobility was slightly affected, but the band shape and pattern were the same. Therefore, it was shown that it can be separated into a clear and sharp band similar to the normal voltage even under high voltage conditions.

Since the gel for electrophoresis can be stably stored for a long period of time by using the gel buffer of the present invention, a uniform and excellent quality precast gel can be produced, and such a precast gel can be produced. Can be produced in large quantities. Since mass production and long-term storage are possible, it is possible to provide the same lot and obtain highly accurate and reproducible data. Furthermore, if the prepared gel is used, high-speed electrophoresis can be performed under high voltage and / or high current conditions, and bands can be separated with high resolution, leading to a reduction in analysis time.

Claims

A gel buffer for electrophoresis,
A buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine;
Glycine and at least one zwitterion with an acid dissociation constant pKa of 9.6 ≦ pKa ≦ 11; and at least one acid;
A gel buffer for electrophoresis, wherein the pH of the gel buffer for electrophoresis is between 5.5 and 7.5.
The gel buffer for electrophoresis according to claim 1, wherein a concentration of the buffer is 50 mM to 300 mM, a concentration of the glycine is 10 mM to 1000 mM, and a concentration of the at least one zwitterion is 10 to 200 mM. liquid.
3. The zwitterion according to claim 1 or 2, wherein the zwitterion is selected from alanine, arginine, cysteine, aspartic acid, glutamic acid, proline, γ-carboxyglutamic acid, γ-aminobutyric acid, 2-aminoisobutyric acid, 6-aminocaproic acid, and CAPS. The gel buffer for electrophoresis as described.
The gel buffer for electrophoresis according to any one of claims 1 to 3, further comprising one or more additives selected from sugars, polyhydric alcohols, and salts.
A polyacrylamide gel for electrophoresis produced using the gel buffer for electrophoresis according to any one of claims 1 to 4.
A buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine; glycine and acid dissociation constant pKa of 9.6 ≦ A polyacrylamide gel for electrophoresis comprising: at least one zwitterion with pKa ≦ 11; and at least one acid.
The polyacrylamide gel for electrophoresis according to claim 5, which is a precast gel.
A method for separating a polypeptide, protein or nucleic acid using the polyacrylamide gel for electrophoresis according to any one of claims 5 to 7.
Acrylamide; crosslinker; buffer selected from tris (hydroxymethyl) aminomethane, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, monoethanolamine, diethanolamine, and triethanolamine; glycine and acid dissociation constant pKa The presence of a polymerization initiator in a condition where the pH is in the range of 5.5 to 7.5, wherein at least one zwitterion with a 9.6 ≦ pKa ≦ 11; and at least one acid; A method for producing a polyacrylamide gel for electrophoresis, which is polymerized under the following conditions.