JP5967751B2 - Precast gel for electrophoresis, production method and use thereof - Google Patents

Description

The present invention relates to a polyacrylamide precast gel for electrophoresis for biochemical drug analysis, a method for using the same, and a method for producing the same.

Polyacrylamide precast gels for electrophoresis are widely used as basic research tools in many fields such as biology, medicine, fisheries, and veterinary medicine for the purpose of detection and quantitative analysis of important substances such as proteins. ing. In particular, since polyacrylamide gel is an artificially synthesized substance, gels with different separation characteristics can be easily prepared by changing the formulation. For this reason, the use of precast gels that are mass-produced to have various separation characteristics in advance greatly reduces the labor of analysis, and contributes to production and quality control in this field because they are uniform and have good reproducibility. In addition, when supplying mass-produced precast gel, it is expected that the storage stability is good.

In recent years, protein modification status has begun to attract attention as an analysis target in the life science field. Proteins are functionalized by more than 300 post-translational modifications in vivo, especially in mammals, where it is said that one third of proteins in the body are in a phosphorylated modified state, and the pathogenesis of various diseases In order to lead to early detection and treatment, the phosphorylation modification state of proteins has been investigated.

As a method for analyzing the phosphorylation state of a protein by gel electrophoresis, a polyacrylamide gel obtained by copolymerizing an acrylamide monomer into which a side chain having a chelating ability is introduced into a compound having an anionic group has been proposed. When this gel is used, the mobility varies depending on the phosphorylation modification state, and even proteins having the same structure can be separated by the phosphorylation state. (Patent Documents 1 and 2)

Conventionally, the combination of buffer solutions proposed by Remley has been widely used for polyacrylamide gel electrophoresis, and has become a common method. The Remley method uses a partially neutralized product of tris (hydroxymethyl) aminomethane (hereinafter abbreviated as “Tris”) with hydrochloric acid as the gel buffer, and Tris and glycine as the electrophoresis buffer (Lemley's electrophoresis buffer). liquid). In the gel buffer in this method, Tris is partially neutralized with hydrochloric acid so as to have a pH of 8.8 (Lemley gel buffer). (Non-Patent Document 1)

In particular, for low molecular weight proteins, a method using Tris or Tricine in an electrophoresis buffer proposed by Shagar et al. Is widely used. In the gel buffer solution in this method, Tris is neutralized with hydrochloric acid so that the pH is 8.45. (Non-Patent Document 2)

Also in Patent Document 1, an example using the Remley method is proposed as an example.

However, at the pH of the gel buffer of Remley or Shagar et al., The amide group undergoes a hydrolysis reaction over time, the migration distance of the protein becomes shorter and the separation image becomes unclear, so the gel cannot be stored for a long time. It was. A precast gel that supplies a mass-produced gel in advance needs to be supplied with a limited shelf life, and it is required that the storage stability is good.

As reported in Patent Document 3, Patent Document 4, and Patent Document 5, the present applicant has also conducted extensive research on quality improvement, production method, and use method of polyacrylamide gel for electrophoresis, and has solved the problem. There is a background.

JP-A-2005-351765 International Publication No. 2007-015312 JP-A-4-184163 JP 2001-159621 A JP 2002-277438 A U. K. Laemmli, Nature 227, 680 (1970) H. Schagger, ANALYTICAL BIOCHEMISTRY 166, 368-379 (1987)

The object of the present invention is to allow the use of Remley or Shagar's running buffer, which has a long-term stable protein separation and gel shape, and is widely used in analysis methods. An object of the present invention is to provide a precast gel for electrophoresis which can be carried out and used for the purpose of analyzing the phosphorylation modification state of a protein.

As a result of intensive studies to solve the above problems, the present inventors have found the following invention.
That is, the invention of claim 1 has an acrylamide copolymer having a structure represented by the following formula (A) in at least a part of the structure and containing a buffer solution having the following compositions (1) and (2). The present invention relates to a precast gel for electrophoresis, which is an aqueous gel.
(1) Tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes as glycine and / or tricine, amphoteric electrolytes other than glycine and / or tricine An amphoteric electrolyte containing threonine as an amphoteric electrolyte other than glycine and / or tricine in an amount of 63.2 to 100 mol% based on glycine and / or tricine.
(2) pH is 6.0-6.8.

Compound (A)
In the formula, M ²⁺ is a transition metal ion.

A second aspect of the present invention, in one or more ampholytes, according to claim 1, the range of a base dissociation constant of glycine and / or ampholytes than tricine is characterized in that it is a 6.6 to 9.6 Precast gel for electrophoresis.

In the invention of claim 3, the concentration of tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane in the gel is 0.07 to 0.2 mol / L. The precast gel for electrophoresis according to claim 1 or 2 , wherein the total concentration of the amphoteric electrolytes of the species or more is 0.1 to 0.5 mol / L.

A fourth aspect of the present invention, acrylamide, the following compound (B), a crosslinking agent, a tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more ampholytes Glycine and / or tricine, threonine as an amphoteric electrolyte other than glycine and / or tricine, and the ampholyte other than glycine and / or tricine is in the range of 63.2 to 100 mol% with respect to glycine and / or tricine. A method for producing a precast gel for electrophoresis, comprising polymerizing a mixed aqueous solution comprising an ampholyte contained therein and having a pH of 6.0 to 6.8.


Compound (B)
In the formula, M ²⁺ is a transition metal ion.

The invention of claim 5 uses a precast gel for electrophoresis according to claims 1 to 3 and a buffer for electrophoresis containing tris (hydroxymethyl) aminomethane in which dodecyl sulfate is present and an amphoteric electrolyte. A method of using a precast gel for electrophoresis, characterized by analyzing a phosphorylated modification state.

According to the present invention, protein separation ability and gel shape are stable for a long period of time, and Remley or Shagar's running buffer, for which analysis methods are widely used, can be used, and the user can perform analysis economically and efficiently. In addition, it is possible to provide a precast gel for electrophoresis used for the purpose of analyzing the phosphorylation modification state of a protein.

The electrophoresis precast gel of the present invention comprises a separation medium for electrophoresis, a gel buffer, and a support for holding the separation medium. The separation medium for electrophoresis of the present invention includes acrylamide, the following compound (B), a crosslinking agent, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more kinds. It can be produced by polymerizing an aqueous mixture solution having a pH of 6.0 to 6.8.

Compound (B)
In the formula, M ²⁺ is a transition metal ion.

The compound (B) is desirably copolymerized in the range of 0.003 to 0.3% by mass with respect to acrylamide. If the amount is more than 0.3% by mass, the degree of polymerization is not sufficiently increased and is not suitable for use as a migration medium, and if it is 0.003% by mass or less, the desired separation effect due to the phosphorylated state cannot be obtained.

The transition metal ion of the compound (B) is preferably a divalent ion such as iron, manganese, copper, or zinc, and particularly preferably a divalent zinc ion. Compound (B) may be mixed with acrylamide in a state where transition metal ions are coordinated, or a divalent transition metal compound may be added during polymerization to form a coordination structure in the copolymer. As the transition metal compound, zinc chloride, zinc acetate, zinc sulfate or the like can be used.

As the crosslinking agent, a general divinyl compound such as N, N-methylenebisacrylamide, N, N-diallyl tartaramide, and the like can be used.

The crosslinking agent is preferably copolymerized in the range of 0.5 to 7% by mass with respect to the total mass of acrylamide and the compound (B). If it is 0.5 mass% or less, the gel becomes too soft and difficult to operate, and if it is 7 mass% or more, a clear separation pattern cannot be obtained.

It is preferable for use as a precast gel for electrophoresis that the total of the acrylamide, the compound (B) and the crosslinking agent is 3 to 25% by mass.

The gel buffer contains tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes.

Glycine can be used as the ampholyte when using the Remley running buffer, and either glycine or tricine can be used when using the Shagar running buffer. Further, by combining glycine and / or tricine with other ampholytes, it is possible to expand the molecular weight range.

The amphoteric electrolyte other than glycine or tricine preferably has a base dissociation constant in the range of 6.6 to 9.6. Specific examples of ampholytes include glycine, tricine, serine, threonine, phenylalanine, glutamic acid, tryptophan, methionine, alanine, valine, aspartic acid,
N, N-bis (2-hydroxyethyl) glycine, tris (hydroxymethyl) methylaminopropanesulfonic acid, 2-aminoethylsulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 3-N-morpholinopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, N-2-hydroxyethylpiperazinepropane There are sulfonic acid, glycylglycine, tris (hydroxymethyl) methylaminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, 2-aminoethylsulfonic acid, etc. It can also be used in combination.

These ampholytes are added to moderate the potential gradient in the gel so that the molecular weight cut-off range is suitable for protein separation and the separation pattern is clear. When electrophoresis is performed using Remley or Shagar's running buffer and a gel with a pH of 6.0 to 6.8, using a gel to which no ampholyte is added, the migration speed is high and the molecular weight range is extremely low. The result is a high molecular region, or the migration speed is slow and the separation pattern is unclear and unsuitable for separation. Particularly preferably, when two or more amphoteric electrolytes are combined, the potential gradient in the gel changes more slowly than the case of glycine or tricine alone, because they move to the anode side in order from the lowest pKb. A clear separation pattern can be obtained in a particularly low-molecular region of possible proteins.

In addition, when the base dissociation constant pKb of the coexisting ampholyte is pKb <6.6, the coexisting ampholyte becomes in an acid dissociation state in the gel, changes the pH of the gel buffer, and acts as a trailing ion. A band that should not be present is detected. In the case of pKb> 9.6, since the base dissociation constant is higher than that of glycine, it does not contribute to the action of moderately changing the potential gradient in the gel.

The concentration of tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane in the gel buffer is 0.07 mol / L to 0.2 mol / L, preferably 0.08 mol / L. L to 0.1 mol / L. This range is a range in which the amount of leading ions in the gel necessary to have a migration time equivalent to that of a gel prepared using the Remley gel buffer can be contained. When the concentration of tris (hydroxymethyl) aminomethane in the gel is lower than 0.07 mol / L, the entire electrophoretic image becomes unclear and becomes unusable for protein separation analysis. When the Tris concentration in the gel is higher than 0.2 mol / L, the leading ion concentration in the gel is increased, the migration time is extended, and the analysis work efficiency is deteriorated.

The total concentration of glycine and / or tricine and the amphoteric electrolyte coexisting with them is preferably 0.1 to 0.5 mol / L. Most preferably, it is 0.1-0.3 mol / L. If the total ampholyte concentration is less than 0.1 mol / L, the effect is small, and the electrophoretic image is unclear and the purpose of use cannot be achieved. You will not be able to get.

The amount of the coexisting amphoteric electrolyte is preferably 0.1 to 100 mol% with respect to glycine and / or tricine. When the addition amount is lower than 0.1 mol%, it does not contribute to the action of gradual change of the potential gradient in the gel, and when it is added more than 100 mol%, the separation pattern becomes unclear or should not be clear but originally A band is detected.

The pH of the gel buffer is 6.0 to 6.8. When the pH is higher than 6.8, hydrolysis of the polyacrylamide gel tends to proceed, and the storage expiration date cannot be prolonged. When the pH is lower than 6.0, the stability as a polyacrylamide gel is good, but the electrophoretic image of the protein is unclear and unusable for practical use. At pH 6.8, refrigerated storage lasts for 6 months, and at pH 6.3, the hydrolysis rate is remarkably reduced compared to pH 6.8 because it is close to the pH of acrylamide itself. As a result, the gel shape and electrophoretic image have not been changed for a long period of one year and are stable.

For pH adjustment, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane are neutralized with hydrochloric acid or acetic acid.

The precast gel of the present invention may contain a water-soluble polymer such as agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, polymethyl vinyl ether, etc. in order to give the gel elasticity and improve gel strength. .

The precast gel of the present invention is prepared by filling a glass or resin carrier with an aqueous solution containing acrylamide, the compound (B), a crosslinking agent and the gel buffer, and then irradiating with a polymerization initiator and / or ultraviolet rays or ionizing. It is produced by gelling with radicals generated by irradiation with actinic radiation. As a polymerization initiator, a redox type that uses a peroxide such as ammonium persulfate (hereinafter abbreviated as APS) and a reducing agent such as N, N, N ′, N′tetramethylethylenediamine (hereinafter abbreviated as TEMED) in combination. A polymerization initiator is awarded, but is not particularly limited. The peroxide and reducing agent are used in an amount of 0.05 to 5% (mass / volume) based on the total monomers. The polymerization temperature is not particularly limited as long as it is a temperature at which the initiator functions, but is usually preferably in the range of 15 to 50 ° C.

The precast gel for electrophoresis of the present invention is used for separation according to the phosphorylation state of a protein using a buffer for electrophoresis containing tris (hydroxymethyl) aminomethane and an amphoteric electrolyte.

The composition of the electrophoresis buffer is preferably an electrophoresis buffer having a composition of Tris 0.025 mol / L, glycine 0.192 mol / L, and SDS 0.1% by weight. This composition is generally used for separation of proteins by gel electrophoresis as a buffer for running Lemmy.

Alternatively, it is desirable to use an electrophoresis buffer having a composition of tris (hydroxymethyl) aminomethane 0.1 mol / L, tricine 0.1 mol / L, and SDS 0.1% by mass. This composition is generally used as a buffer for electrophoresis of a sugar, particularly for the separation of low molecular weight proteins by gel electrophoresis.

(Example)
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.

Preparation of precast gel for electrophoresis Silicon seal to prevent leakage of 1 mm spacer and monomer liquid between a rectangular glass plate with a width of 12 cm and a length of 10 cm and a glass plate of the same size with a concave cut at the top Assemble the glass plate. Acrylamide concentration of 12% T, 0.03% by mass of the compound (B) with respect to acrylamide (using zinc as a transition metal), N, N-methylenebisacrylamide concentration of 3% C, and Example-1 described in Table-1 After adding and mixing to a monomer solution containing a gel buffer of the composition of APS 0.002 mol / L and TEMED 0.006 mol / L, the mixture was injected into a plate and polymerized at 25 ° C. A separation layer of acrylamide gel was obtained.

Furthermore, for a monomer solution containing an acrylamide concentration of 5% T, an N, N-methylenebisacrylamide concentration of 3% C, and a gel buffer solution having the composition described in Table 1, APS 0.002 mol / L and TEMED 0.006 mol / After adding and mixing so as to be L, the mixture was poured onto the separation layer and polymerized at 25 ° C. to obtain a concentrated layer of polyacrylamide gel for electrophoresis.

Electrophoresis test method Electrophoresis was performed using an electrophoresis buffer of Remley formulation of Tris 0.025 mol / L, glycine 0.192 mol / L, and SDS 0.1% by mass. Α-casein and dephosphorylated α-casein were electrophoresed as protein samples. Dephosphorylated α-casein was dephosphorylated by adding alkaline phosphatase (manufactured by Nippon Gene) to the α-casein solution and allowing it to stand overnight.
Electrophoresis was performed at a constant current of 20 mA, and energization was stopped when the migration terminal was 10 mm from the bottom.
Staining was performed in a 0.05% by weight Coomassie brilliant blue (hereinafter abbreviated as CBB) G-250, 12% by volume acetic acid, 30% by volume methanol solution for 60 minutes, and decolorization was 12% by volume. The reaction was carried out in acetic acid and 15% by volume methanol solution for 120 minutes using a shaker.
Table 2-1 shows the mobility of α-casein and dephosphorylated α-casein and their differences and the sharpness of each band.
Mobility refers to the distance traveled by a protein in percent.
(Mobility%) = (Distance from bottom of well to each band position)
/ (Distance from bottom of well to end of migration) × 100

A precast gel prepared by the same method as in Example-1 was stored for 8 months under refrigeration, and then a migration test was performed in the same manner as in Example-1. The results are shown in Example-2 in Table 2-1.

A migration test was performed in the same manner as in Example 1 except that a precast gel was prepared using the gel buffer of Example-2 in Table 1. The results are shown in Example-2 in Table 2-1.

The migration test was carried out in the same manner as in Example 1 except that the pre-cast gel was prepared using the gel buffer of Example-3 in Table 1 with the acrylamide concentration changed to 7.5% T. The results are shown in Example 3 in Table 2-1.

A precast gel was prepared in the same manner as in Example-1 except that the gel buffer of Example-4 in Table 1 was used. Electrophoresis was performed using a running buffer of a Shagar formulation of Tris 0.1 mol / L, Tricine 0.1 mol / L, SDS 0.1 mass%. The protein sample and detection method were the same as in Example 1. The results are shown in Example 4 in Table 2-1.

(Comparative Example 1)
Implemented except that a precast gel was prepared using 0.375 mol / L Tris-HCl gel buffer solution at pH 8.8 for the separation layer and 0.125 mol / L Tris-HCl gel buffer solution at pH 6.8 for the concentration layer. The migration test was performed in the same manner as in Example-1. The results are shown in Comparative Example-1 in Table 2-2.

(Comparative Example-2)
A precast gel prepared in the same manner as in Comparative Example-1 was stored for 6 months under refrigeration, and then a migration test was performed in the same manner as in Example-1. The results are shown in Comparative Example-2 in Table 2-2.

(Comparative Example-3)
A precast gel was prepared using 0.375 mol / L Tris-hydrochloric acid gel buffer solution at pH 6.8 in the separation layer and 0.125 mol / L Tris-hydrochloric acid gel buffer gel buffer solution in the concentration layer at pH 6.8. Except for the above, the migration test was performed in the same manner as in Example-1. The results are shown in Comparative Example-3 in Table 2-2.

(Comparative Example-4)
A precast gel was prepared in the same manner as in Example 1 except that the compound (B) was not added, and a migration test was performed. The results are shown in Comparative Example-4 in Table 2-2.

(Table 1) Gel buffer composition
MOPS: 3-N-morpholinopropanesulfonic acid HEPES: N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

(Table 2-1) Mobility (%)

(Table 2-2) Mobility (%)

As shown in Table 2-1, in the precast gels of Examples-1 and 3 to 5, phosphorylated α-casein and dephosphorylated α-casein are both clear bands and a clear mobility difference can be obtained. The precast gel was able to separate proteins according to their phosphorylation state using Remley or Shagar's running buffer.

As shown in Table 2-1, from Example-2, even when the precast gel for electrophoresis of the present invention has been produced for 6 months, the same band sharpness, mobility, and mobility difference due to phosphorylation state as those immediately after the production were obtained. I was able to get it.

As shown in Table 2-2, in the precast gels of Comparative Examples-1 and 2 using the gel buffer of the Remley formulation, a clear mobility difference could be obtained immediately after the preparation, but phosphorylated-dephosphorylated protein. The difference in mobility was smaller than that of Example 1, and a clear band could not be obtained after 6 months from the preparation.

As shown in Table 2-2, in the precast gel of Comparative Example-3, a clear mobility difference could not be obtained. The band was also unclear.

As shown in Table 2-2, in the precast gel of Comparative Example-4, a clear band could be obtained, but a clear mobility difference could not be obtained between α-casein and dephosphorylated α-casein. .

From the above results, it is clear that the precast gel for electrophoresis of the present invention can clearly separate the phosphorylated state of the protein and has the long-term stability required for the precast gel.

By using the precast gel for electrophoresis of the present invention, the separation ability of the protein and the shape of the gel are stable for a long period of time, and it is possible to use the Remley or Shagar electrophoresis buffer which is widely used for the analysis method. In particular, it is possible to analyze the phosphorylation modification state of a protein, and its industrial applicability is very large.

Claims (5)

It is an acrylamide copolymer aqueous gel having a structure represented by the following formula (A) in at least a part of the structure and containing a buffer solution having the following compositions (1) and (2). Precast gel for electrophoresis.
(1) Tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes as glycine and / or tricine, amphoteric electrolytes other than glycine and / or tricine An amphoteric electrolyte containing threonine as an amphoteric electrolyte other than glycine and / or tricine in an amount of 63.2 to 100 mol% based on glycine and / or tricine.
(2) pH is 6.0-6.8.

Structural unit (A)
In the formula, M ²⁺ is a transition metal ion. 2. The precast gel for electrophoresis according to claim 1 , wherein the base dissociation constant of the ampholytes other than glycine and / or tricine is 6.6 to 9.6 in one or more ampholytes. The concentration of tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane in the gel is 0.07 to 0.2 mol / L, and the concentration of one or more ampholytes claim 1 electrophoresis precast gels according to 2, wherein the total is 0.1 to 0.5 mol / L. Acrylamide, the following compound (B), a cross-linking agent, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more ampholytes as glycine and / or tricine, Threonine is contained as an amphoteric electrolyte other than glycine and / or tricine, and the ampholyte other than glycine and / or tricine is comprised of an amphoteric electrolyte in a range of 63.2 to 100 mol% with respect to glycine and / or tricine , A method for producing a precast gel for electrophoresis, comprising polymerizing a mixture aqueous solution having a pH of 6.0 to 6.8.

Compound (B)
Where M2 + is a transition metal ion Using the electrophoresis precast gel according to claims 1 to 3 and a buffer for electrophoresis containing tris (hydroxymethyl) aminomethane containing dodecyl sulfate and an ampholyte, the phosphorylation modification state of the protein is analyzed. The use method of the precast gel for electrophoresis characterized by the above-mentioned.