WO2013089076A1 - Precast gel for electrophoresis, method for producing same, and method for using same - Google Patents

Precast gel for electrophoresis, method for producing same, and method for using same Download PDF

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WO2013089076A1
WO2013089076A1 PCT/JP2012/081997 JP2012081997W WO2013089076A1 WO 2013089076 A1 WO2013089076 A1 WO 2013089076A1 JP 2012081997 W JP2012081997 W JP 2012081997W WO 2013089076 A1 WO2013089076 A1 WO 2013089076A1
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gel
electrophoresis
hydroxymethyl
acid
tris
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PCT/JP2012/081997
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French (fr)
Japanese (ja)
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由希子 峰岸
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ハイモ株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • 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 constituting the living body such as proteins. ing.
  • polyacrylamide gel is an artificially synthesized substance, gels with different separation characteristics can be easily prepared by changing the formulation.
  • 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.
  • it is expected that the storage stability is good.
  • 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.
  • Non-Patent Document 2 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.
  • Patent Document 1 an example using the Remley method is proposed as an example.
  • Patent Document 4 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.
  • 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.
  • the present invention includes 1) an aqueous 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). It is related with the precast gel for electrophoresis characterized by being a gel. (1) Tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes. (2) pH is 6.0 to 6.8. Compound (A) In the formula, M 2+ is a transition metal ion.
  • the invention according to claim 2 is the precast gel for electrophoresis according to claim 1, wherein the one or more ampholytes in the gel buffer essentially comprise glycine and / or tricine.
  • the invention of claim 3 is characterized in that in one or more ampholytes, the range of the base dissociation constant of the ampholytes other than glycine and / or tricine is 6.6 to 9.6.
  • the ampholyte other than glycine and / or tricine is 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 It is at least one selected from sulfonic acid, glycylglycine, tris (hydroxymethyl) methylamin
  • the invention of claim 5 is characterized in that the ampholyte other than glycine and / or tricine is contained in a range of 0.1 to 100 mol% with respect to glycine and / or tricine. Precast gel for electrophoresis.
  • 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 invention of claim 7 includes acrylamide, the following compound (B), a crosslinking agent, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more ampholytes.
  • a method for producing a precast gel for electrophoresis comprising 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 invention of claim 8 uses the precast gel for electrophoresis according to claims 1 to 6 and a buffer for electrophoresis containing tris (hydroxymethyl) aminomethane in which dodecyl sulfate is present and an ampholyte.
  • the resolution 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's running buffer, which is widely used for analysis, and the user can perform the analysis economically and efficiently.
  • 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 a mixed aqueous solution comprising an amphoteric electrolyte having a pH of 6.0 to 6.8.
  • M 2+ is a transition metal ion.
  • the compound (B) is preferably 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.
  • 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.
  • crosslinking agent general divinyl compounds such as N, N-methylenebisacrylamide and N, N-diallyltartaramide may 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 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.
  • 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.
  • amphoteric electrolyte other than glycine or tricine preferably has a base dissociation constant in the range of 6.6 to 9.6.
  • 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-hydroxyethylpiperazinepropan
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ampholyte coexisting with them is preferably 0.1 to 0.5 mol / L. Particularly preferred is 0.1 to 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 ampholyte is preferably 0.1 to 100 mol% with respect to glycine and / or tricine.
  • the addition amount is lower than 0.1 mol%, it does not contribute to the action of slowing the change in 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.
  • hydrolysis of the polyacrylamide gel tends to proceed, and the storage expiration date cannot be prolonged.
  • 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.
  • 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.
  • 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, and polymethyl vinyl ether in order to give the gel elasticity and improve gel strength. .
  • a water-soluble polymer such as agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, and polymethyl vinyl ether
  • 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 subjecting the polymerization initiator and / or ultraviolet irradiation or ionization to the gel. It is produced by gelation with radicals generated by irradiation with sexual radiation.
  • 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).
  • 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 sodium dodecyl sulfate (hereinafter abbreviated as SDS) 0.1% by mass. .
  • SDS sodium dodecyl sulfate
  • 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.
  • Preparation method 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 12% by mass (acrylamide + crosslinking agent, total concentration)
  • Compound (B) 0.03% by mass of acrylamide (using zinc as transition metal), 3% by mass of N, N-methylenebisacrylamide (vs.
  • a monomer containing an acrylamide concentration of 5% by mass (acrylamide + crosslinking agent, total concentration), N, N-methylenebisacrylamide 3% by mass (based on acrylamide concentration), and a gel buffer having the composition described in Table 1.
  • the solution was injected onto the separation layer and polymerized at 25 ° C. to obtain a concentrated layer of polyacrylamide gel for electrophoresis. .
  • Electrophoresis test method Electrophoresis was carried out using an electrophoresis buffer of Remley formulation of Tris 0.025 mol / L, glycine 0.192 mol / L, and SDS 0.1% by mass. ⁇ -casein and dephosphorylated ⁇ -casein were electrophoresed as protein samples. Dephosphorylated ⁇ -casein was dephosphorylated by adding alkaline phosphatase (manufactured by Nippon Gene) to the ⁇ -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.
  • alkaline phosphatase manufactured by Nippon Gene
  • Example 2 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.
  • Example 3 An electrophoresis test was performed in the same manner as in Example 1 except that a precast gel was prepared using the gel buffer of Example 3 in Table 1. The results are shown in Example 3 in Table 2-1.
  • Example 4 The migration test was performed in the same manner as in Example-1, except that the acrylamide concentration was changed to 7.5% (acrylamide + crosslinking agent vs. total concentration) and the precast gel was prepared using the gel buffer of Example-4 in Table 1. Carried out. The results are shown in Example 4 in Table 2-1.
  • a precast gel was prepared in the same manner as in Example 1 except that the gel buffer of Example-5 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-5 in Table 2-1.
  • Comparative Example 1 (Comparative Example 1) Performed except that a precast gel was prepared using 0.375 mol / L Tris-hydrochloric acid gel buffer pH 8.8 for the separation layer and 0.125 mol / L Tris-hydrochloric acid gel buffer pH 6.8 for the concentration layer. A migration test was performed 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-4 A precast gel was prepared and a migration test was conducted in the same manner as in Example 1 except that the compound (B) was not added. The results are shown in Comparative Example-4 in Table 2-2.
  • phosphorylated ⁇ -casein and dephosphorylated ⁇ -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.
  • 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.
  • 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.

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Abstract

The purpose of the present invention is to provide a precast gel for electrophoresis, which can be used for analyzing the state of phosphorylation modification of a protein, can use a Laemmli's or Schagger's electrophoresis buffer solution for which the analysis method has been in widespread use, and can be used by a user ecomonically and efficiently. In the present invention, the purpose can be achieved by a precast gel for electrophoresis, characterized by being an aqueous acrylamide copolymer gel that has a specific structure in at least a part of the structure thereof and contains a buffer solution having the composition (1) and the property (2): (1) tris(hydroxymethyl)aminomethane and/or bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, and at least one amphoteric electrolyte; and (2) a pH value of 6.0 to 6.8.

Description

電気泳動用プレキャストゲル、その製造方法および使用方法Precast gel for electrophoresis, production method and use thereof
本発明は生化学医薬分析を目的とする電気泳動用ポリアクリルアミドプレキャストゲル、その使用法及びその製法に関するものである。 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.
電気泳動用ポリアクリルアミドプレキャストゲルは、蛋白質など生体を構成する重要な物質の検出並びに定量分析を目的として、生物学、医学、水産学、獣医学など多くの分野における基礎的研究手段とし広く使用されている。特にポリアクリルアミドゲルは人工的に合成された物質であるため、処方を変えることで分離特性の異なるゲルを容易に作成できる。そのため、予め様々な分離特性を持つように量産されたプレキャストゲルを使用することで分析の手間を大幅に省き、均一で再現性が良いことから当該分野における生産および品質管理に貢献するところが大きい。また、量産されたプレキャストゲルを供給するに際しては、保存安定性が良好であることが期待される。 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 constituting the living body 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.
ライフサイエンス分野の分析対象としては、近年は蛋白質の修飾状態が注目され始めている。蛋白質は、生体内で３００種類以上の翻訳後修飾によって機能を付与されており、特に、哺乳類は生体内の３分の１の蛋白質がりん酸化修飾状態にあるといわれ、様々な疾患の発症機構や早期発見、治療につなげるため、蛋白質のりん酸化修飾状態を調べることが行われている。 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.
ゲル電気泳動法による蛋白質のりん酸化状態の分析方法としてアニオン性基をもつ化合物に対しキレート形成能力を持つ側鎖を導入したアクリルアミドモノマーを共重合したポリアクリルアミドゲルが提案されている。このゲルを用いると、りん酸化修飾状態により移動度が異なり、同一構造の蛋白質でもりん酸化状態により分離することが可能となる。（特許文献１、２） 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)
従来、ポリアクリルアミドゲル電気泳動法には、レムリーによって提案された緩衝液の組み合わせが広く使用され、一般的な方法となっている。レムリーの方法はゲル緩衝液としてトリス(ヒドロキシメチル)アミノメタン(以下トリスと略す)の塩酸による部分中和物を使用し、泳動緩衝液にはトリス、グリシンを使用している（レムリーの泳動緩衝液）。この方法におけるゲル緩衝液は、ｐＨが８．８になるようにトリスが塩酸により部分中和されている（レムリーのゲル緩衝液）。（非特許文献１） 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)
特に、低分子量の蛋白質に対しては、特にシャガーらが提案した泳動緩衝液にトリス、トリシン使用した方法が広く利用されている。この方法におけるゲル緩衝液は、pＨが８．４５になるようにトリスが塩酸により中和されている。（非特許文献２） 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)
前記特許文献１においても、レムリーの方法を用いた例が実施例として提案されている。 Also in Patent Document 1, an example using the Remley method is proposed as an example.
しかしレムリーあるいはシャガーらのゲル緩衝液のpHでは、アミド基は経時的に加水分解反応を受け、蛋白質の泳動距離が短くなると共に分離像は不鮮明となるのでゲルを長期に保存することはできなかった。予め量産したゲルを供給するプレキャストゲルにおいては保管期限を限定して供給する必要があり保存安定性が良好であることが必要とされる。 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.
本出願人においても特許文献３及び特許文献４、特許文献５に報告されているように、電気泳動用ポリアクリルアミドゲルの品質向上や製造方法、使用方法について鋭意研究を行い、課題を解決してきた経緯がある。 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.
特開２００５－３５１７６５号公報JP 2005-351765 A 国際公開２００７－０１５３１２号公報International Publication No. 2007-015312 特開平４－１８４１６３号公報JP-A-4-184163 特開２００１－１５９６２１号公報JP 2001-159621 A 特開２００２－２７７４３８号公報JP 2002-277438 A
本発明の目的は、蛋白質の分離能及びゲルの形状が長期安定で、分析方法が普及しているレムリーあるいはシャガーの泳動緩衝液が使用可能であり、使用者が経済的かつ効率的に分析を遂行することが可能な、蛋白質のりん酸化修飾状態の分析を目的として使用される電気泳動用プレキャストゲルを提供することにある。 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.
本発明者らは、前記課題を解決するために鋭意研究を重ねた結果、以下の発明を見出した。
すなわち本発明は、１）その構造中の少なくとも一部に下記式（Ａ）で表される構造を有し、下記組成（１）および（２）の緩衝液を含有するアクリルアミド系共重合体水性ゲルであることを特徴とする電気泳動用プレキャストゲルに関する。
（１）トリス(ヒドロキシメチル)アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンと1種以上の両性電解質。
（２）ｐＨが６．０～６．８。
Figure JPOXMLDOC01-appb-C000003
  
化合物（Ａ）
式中、Ｍ２+は遷移金属イオン　 As a result of intensive studies to solve the above problems, the present inventors have found the following invention.
That is, the present invention includes 1) an aqueous 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). It is related with the precast gel for electrophoresis characterized by being a gel.
(1) Tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes.
(2) pH is 6.0 to 6.8.
Figure JPOXMLDOC01-appb-C000003
Compound (A)
In the formula, M 2+ is a transition metal ion.
請求項２の発明は、ゲル緩衝液中の１種以上の両性電解質が、グリシンおよび／またはトリシンを必須とすることを特徴とする請求項１に記載の電気泳動用プレキャストゲル。 The invention according to claim 2 is the precast gel for electrophoresis according to claim 1, wherein the one or more ampholytes in the gel buffer essentially comprise glycine and / or tricine.
請求項３の発明は、１種以上の両性電解質において、グリシンおよび／またはトリシン以外の両性電解質の塩基解離定数の範囲が６．６～９．６であることを特徴とする請求項１あるいは２に記載の電気泳動用プレキャストゲル。 The invention of claim 3 is characterized in that in one or more ampholytes, the range of the base dissociation constant of the ampholytes other than glycine and / or tricine is 6.6 to 9.6. A precast gel for electrophoresis as described in 1.
請求項４の発明は、グリシンおよび／またはトリシン以外の両性電解質が、セリン、スレオニン、フェニルアラニン、グルタミン酸、トリプトファン、メチオニン、アラニン、バリン、アスパラギン酸、
Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン、トリス（ヒドロキシメチル)メチルアミノプロパンスルホン酸、２－アミノエチルスルホン酸、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）－２－アミノエタンスルホン酸、３－Ｎ－モルホリノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジン－Ｎ‘－２－エタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジンプロパンスルホン酸、グリシルグリシン、トリス（ヒドロキシメチル）メチルアミノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、２－アミノエチルスルホン酸から選択される一種以上であることを特徴とする請求項３に記載の電気泳動用プレキャストゲル。 In the invention of claim 4, the ampholyte other than glycine and / or tricine is 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 It is at least one selected from sulfonic acid, glycylglycine, tris (hydroxymethyl) methylaminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, and 2-aminoethylsulfonic acid. Electrophoretic precast according to claim 3 Gel.
請求項５の発明は、グリシンおよび／またはトリシン以外の両性電解質が、グリシンおよび／またはトリシンに対して０．１～１００ｍｏｌ％の範囲で含まれることを特徴とする請求項１～４に記載の電気泳動用プレキャストゲル。 The invention of claim 5 is characterized in that the ampholyte other than glycine and / or tricine is contained in a range of 0.1 to 100 mol% with respect to glycine and / or tricine. Precast gel for electrophoresis.
請求項６の発明は、ゲル中のトリス（ヒドロキシメチル）アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンの濃度が、０．０７～０．２ｍｏｌ／Ｌであり、１種以上の両性電解質の濃度の合計が０．１～０．５ｍｏｌ／Ｌであることを特徴とする請求項１～５に記載の電気泳動用プレキャストゲル。 In the invention of claim 6, 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 any one of claims 1 to 5, wherein the total concentration of the ampholyte of the species or more is 0.1 to 0.5 mol / L.
請求項７の発明は、アクリルアミド、下記化合物（Ｂ）、架橋剤、トリス(ヒドロキシメチル)アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン、および1種以上の両性電解質からなり、ｐＨが６．０～６．８である混合物水溶液を重合することを特徴とする電気泳動用プレキャストゲルの製造方法。
Figure JPOXMLDOC01-appb-C000004
  
化合物（Ｂ）
式中、Ｍ２+は遷移金属イオン The invention of claim 7 includes acrylamide, the following compound (B), a crosslinking agent, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more ampholytes. A method for producing a precast gel for electrophoresis, comprising polymerizing an aqueous mixture solution having a pH of 6.0 to 6.8.
Figure JPOXMLDOC01-appb-C000004
Compound (B)
In the formula, M 2+ is a transition metal ion.
請求項８の発明は、請求項１～６に記載の電気泳動用プレキャストゲルと、ドデシル硫酸塩が存在するトリス(ヒドロキシメチル)アミノメタン及び両性電解質を含有する泳動用緩衝液を用い、蛋白質のりん酸化修飾状態を分析することを特徴とする電気泳動用プレキャストゲルの使用方法。 The invention of claim 8 uses the precast gel for electrophoresis according to claims 1 to 6 and a buffer for electrophoresis containing tris (hydroxymethyl) aminomethane in which dodecyl sulfate is present and an ampholyte. A method of using a precast gel for electrophoresis, characterized by analyzing a phosphorylated modification state.
本発明により、蛋白質の分離能及びゲルの形状が長期安定で、分析方法が普及しているレムリーあるいはシャガーの泳動緩衝液が使用でき使用者が経済的かつ効率的に分析を遂行することが可能な、蛋白質のりん酸化修飾状態の分析を目的として使用される電気泳動用プレキャストゲルを提供することができる。 According to the present invention, the resolution 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's running buffer, which is widely used for analysis, and the user can perform the 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.
本発明の電気泳動用プレキャストゲルには、泳動用分離媒体とゲル緩衝液および分離媒体を保持するための担持体から構成される。また本発明の前記泳動用分離媒体は、アクリルアミド、下記化合物（Ｂ）、架橋剤、トリス(ヒドロキシメチル)アミノメタンおよび／またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン、および1種以上の両性電解質からなり、ｐＨが６．０～６．８である混合物水溶液を重合することにより製造可能である。 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 a mixed aqueous solution comprising an amphoteric electrolyte having a pH of 6.0 to 6.8.
Figure JPOXMLDOC01-appb-C000005
  
化合物（Ｂ）
式中、Ｍ２+は遷移金属イオン　
Figure JPOXMLDOC01-appb-C000005
Compound (B)
In the formula, M 2+ is a transition metal ion.
前記化合物（Ｂ）はアクリルアミドに対し、０．００３～０．３質量％の範囲で共重合させることが望ましい。０．３質量%よりも多いと重合度が十分に大きくならず泳動用媒体としての使用に適さず、０．００３質量％以下では目的とするりん酸化状態による分離効果が得られない。 The compound (B) is preferably 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.
前記化合物（Ｂ）の遷移金属イオンとしては、鉄、マンガン、銅、亜鉛などの二価イオンが望ましく、特に好ましくは２価亜鉛イオンである。化合物(Ｂ)は遷移金属イオンが配位した状態でアクリルアミドと混合しても良いし、重合時に二価の遷移金属化合物を添加して共重合体中に配位構造を形成しても良い。遷移金属化合物としては、塩化亜鉛、酢酸亜鉛、硫酸亜鉛などを用いることができる。 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.
前記架橋剤としては、Ｎ，Ｎ－メチレンビスアクリルアミド、Ｎ，Ｎ－ジアリル酒石酸アミド等の一般的なジビニル化合物を使用することができる。 As the crosslinking agent, general divinyl compounds such as N, N-methylenebisacrylamide and N, N-diallyltartaramide may be used.
前記架橋剤はアクリルアミドと化合物（Ｂ）の合計質量に対し、０．５～７質量％の範囲で共重合させることが望ましい。０．５質量％以下では、ゲルが柔らかくなりすぎて操作が難しく、７質量％以上では鮮明な分離パターンを得ることが出来ない。 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 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.
前記アクリルアミドと化合物（Ｂ）と架橋剤との合計が３～２５質量％となるように用いることが電気泳動用プレキャストゲルとしての使用に好ましい。 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.
前記ゲル緩衝液は、トリス（ヒドロキシメチル）アミノメタンおよび／またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンと１種以上の両性電解質を含有する。 The gel buffer contains tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and one or more ampholytes.
レムリーの泳動緩衝液を用いる場合には上記両性電解質として、グリシンがあげられ、シャガーの泳動緩衝液を用いる場合にはグリシンまたはトリシンのいずれも用いることができる。またグリシンおよび／またはトリシンとそれ以外の両性電解質を組み合わせることで、分画分子量範囲を広げることが可能となる。 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.
グリシンあるいはトリシン以外の両性電解質は、塩基解離定数の範囲が６．６～９．６であることが好ましい。具体的に両性電解質としてあげれば、グリシン、トリシン、セリン、スレオニン、フェニルアラニン、グルタミン酸、トリプトファン、メチオニン、アラニン、バリン、アスパラギン酸、
Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン、トリス（ヒドロキシメチル)メチルアミノプロパンスルホン酸、２－アミノエチルスルホン酸、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）－２－アミノエタンスルホン酸、３－Ｎ－モルホリノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジン－Ｎ‘－２－エタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジンプロパンスルホン酸、グリシルグリシン、トリス（ヒドロキシメチル）メチルアミノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、２－アミノエチルスルホン酸などあり、これら両性電解質を二種以上組み合わせて用いることもできる。 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.
これらの両性電解質は、ゲル内の電位勾配を緩やかにして分画分子量範囲を蛋白質の分離に適した範囲とし、分離パターンを鮮明にするために添加される。レムリーあるいはシャガーの泳動緩衝液とｐHが６．０～６．８のゲルを用いて電気泳動を行った場合、両性電解質が未添加のゲルを用いると、泳動速度が早く分画分子量範囲が極端に高分子領域になるか、あるいは泳動速度が遅く分離パターンが不鮮明で分離に適さない結果となる。特に好ましくは、２種以上の両性電解質を組み合わせた場合で、ｐＫｂの低いものから順に陽極側へ移動するためグリシンあるいはトリシン単独の場合よりも、ゲル内の電位勾配の変化が緩やかなものとなり分離可能な蛋白質の特に低分子領域で鮮明な分離パターンを得ることができる。 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.
また、共存両性電解質の塩基解離定数ｐＫｂがｐＫｂ＜６．６の場合、共存両性電解質はゲル内で酸解離状態となりゲル緩衝液のｐＨを変化させ、トレイリングイオンとして作用するために分離パターンに本来ないはずのバンドが検出されることになる。ｐＫｂ＞９．６の場合、グリシンよりも塩基解離定数が高いことになるため、ゲル内の電位勾配の変化を緩やかにする作用に寄与しない。 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.
ゲル緩衝液中のトリス（ヒドロキシメチル）アミノメタンおよび／またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンの濃度は、０．０７ｍｏｌ／Ｌ～０．２ｍｏｌ／Ｌ、好ましくは０．０８ｍｏｌ／Ｌ～０．１ｍｏｌ／Ｌである。この範囲は、レムリーのゲル緩衝液を使用して作成したゲルと同等の泳動時間を有するために必要なゲル中のリーディングイオンの量を含有させることのできる範囲である。ゲル中のトリス（ヒドロキシメチル）アミノメタンの濃度が０．０７ｍｏｌ／Ｌよりも低い場合、泳動像全体が不鮮明となり蛋白質の分離分析に使用しがたいものとなる。ゲル中のトリス濃度が０．２ｍｏｌ／Ｌより高くなるとゲル中のリーディングイオン濃度が高くなり、泳動時間が延長し、分析の作業効率が悪くなる。 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.
また、グリシンおよび／またはトリシンと、それらと共存する両性電解質の濃度の合計は０．１～０．５ｍｏｌ／Ｌであることが好ましい。特に好ましくは０．１～０．３ｍｏｌ／Ｌである。全両性電解質濃度が０．１ｍｏｌ／Ｌよりも少なければその効果は小さくなり泳動像は不鮮明で使用目的を達成できないし、０．５ｍｏｌ／Ｌより多くなると、十分な蛋白質のりん酸化状態に対する分離能を得ることができなくなる。 The total concentration of glycine and / or tricine and the ampholyte coexisting with them is preferably 0.1 to 0.5 mol / L. Particularly preferred is 0.1 to 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.
また、共存両性電解質の添加量はグリシンおよび／またはトリシンに対して０．１～１００ｍｏｌ％が好ましい。０．１ｍｏｌ％より低添加量の場合、ゲル内の電位勾配の変化を緩やかにする作用に寄与せず、１００ｍｏｌ％よりも多く添加すると分離パターンは、不鮮明となるか、鮮明でも本来ないはずのバンドが検出される。 The amount of the coexisting ampholyte 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 slowing the change in 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.
前記ゲル緩衝液のｐＨは、６．０～６．８である。ｐＨ６．８よりも高い場合、ポリアクリルアミドゲルの加水分解が進行し易くなり、保管有効期限を長く持たせることが出来ない。ｐＨ６．０より低い場合
、ポリアクリルアミドゲルとしての安定性は良好であるが、蛋白質の泳動像が不鮮明で実用に耐えないものとなる。ｐＨ６．８では冷蔵保存で６ヶ月もち、さらにｐＨ６．３ではアクリルアミド自体のｐＨに近いため、ｐＨ６．８に比べ加水分解速度が著しく減少する。その結果1年もの長期間ゲルの形状、泳動像共に変化は見られず安定である。 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.
ｐＨ調製のためにトリス（ヒドロキシメチル）アミノメタンおよび／またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンは、塩酸または酢酸によって中和される。 For pH adjustment, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane are neutralized with hydrochloric acid or acetic acid.
本発明のプレキャストゲルには、ゲルに弾力性を持たせゲル強度を向上させるため、アガロース、ポリアクリルアミド、ポリビニルアルコール、ポリビニルピロリドン、ポリエチレンオキサイド、ポリメチルビニルエーテル等の水溶性ポリマーを含有させることもできる。 The precast gel of the present invention may contain a water-soluble polymer such as agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide, and polymethyl vinyl ether in order to give the gel elasticity and improve gel strength. .
本発明のプレキャストゲルは、アクリルアミド、前記化合物（Ｂ）、架橋剤および前記ゲル緩衝液を含有する水溶液を、ガラスあるいは樹脂製の担持体に充填した後、重合開始剤および／または紫外線照射あるいは電離性放射線の照射により発生するラジカルによってゲル化させることにより製造する。重合開始剤としては、過硫酸アンモニウム(以下、ＡＰＳと略す)等の過酸化物と、Ｎ，Ｎ，Ｎ’， Ｎ’テトラメチルエチレンジアミン（以下ＴＥＭＥＤと略す）等の還元剤を併用するレドックス型の重合開始剤が使用されるが、これら特に限定されるものではない。上記過酸化物及び還元剤は全モノマーに対し、０．０５～５％(質量／容量）が使用される。また、重合温度は開始剤が機能する温度であれば特に限定されないが、通常１５～５０℃の範囲が好ましい。 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 subjecting the polymerization initiator and / or ultraviolet irradiation or ionization to the gel. It is produced by gelation with radicals generated by irradiation with sexual 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). Although a polymerization initiator is used, these are 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.
本発明の電気泳動用プレキャストゲルは、トリス（ヒドロキシメチル）アミノメタン及び両性電解質を含有する泳動用緩衝液を使用して蛋白質のりん酸化状態に応じた分離をするために使用される。 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.
前記泳動緩衝液の組成は、トリス０．０２５ｍｏｌ／Ｌ、グリシン０．１９２ｍｏｌ／Ｌ、ドデシル硫酸ナトリウム（以下、ＳＤＳと略す）０．１質量％である組成の泳動緩衝液を使用することが望ましい。この組成は、レムリーの泳動用緩衝液として、ゲル電気泳動法による蛋白質の分離に一般的に用いられるものである。 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 sodium dodecyl sulfate (hereinafter abbreviated as SDS) 0.1% by mass. . This composition is generally used for separation of proteins by gel electrophoresis as a buffer for running Lemmy.
あるいは、前記泳動緩衝液の組成は、トリス（ヒドロキシメチル）アミノメタン０．１ｍｏｌ／Ｌ、トリシン０．１ｍｏｌ／Ｌ、ＳＤＳ０．１質量％である組成の泳動緩衝液を使用することが望ましい。この組成は、シャガーの泳動用緩衝液として、特にゲル電気泳動法による低分子量の蛋白質の分離に一般的に用いられるものである。 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.
以下、実施例により本発明を説明するが、本発明はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
泳動用プレキャストゲルの作成方法
  横幅１２ｃｍ、縦１０ｃｍの長方形のガラス板と、上部に凹状の切り込みの入った同寸法のガラス板の間に、厚さ１ｍｍのスペーサーとモノマー液が漏れないようにシリコンのシールを挟みガラスプレートを組み立てる。アクリルアミド濃度１２質量％（アクリルアミド＋架橋剤、対全量濃度）、前記化合物（Ｂ）対アクリルアミド０．０３質量％（遷移金属として亜鉛を使用）、Ｎ，Ｎ－メチレンビスアクリルアミド３質量％（対アクリルアミド濃度）、並びに表－１に記載する実施例－1の組成のゲル緩衝液を含有するモノマー溶液に対し、ＡＰＳ０．００２ｍｏｌ／ＬおよびＴＥＭＥＤ０．００６ｍｏｌ／Ｌとなるように添加混合後、プレート内に注入して２５℃下で重合させ、電気泳動用ポリアクリルアミドゲルの分離層を得た。 Preparation method 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: 12% by mass (acrylamide + crosslinking agent, total concentration), Compound (B): 0.03% by mass of acrylamide (using zinc as transition metal), 3% by mass of N, N-methylenebisacrylamide (vs. acrylamide) Concentration), and a monomer solution containing the gel buffer having the composition of Example-1 described in Table 1, was added and mixed so as to be APS 0.002 mol / L and TEMED 0.006 mol / L, and then added to the plate. The mixture was injected and polymerized at 25 ° C. to obtain a polyacrylamide gel separation layer for electrophoresis.
さらに、アクリルアミド濃度５質量％（アクリルアミド＋架橋剤、対全量濃度）、Ｎ，Ｎ－メチレンビスアクリルアミド３質量％（対アクリルアミド濃度）、並びに表－１に記載する組成のゲル緩衝液を含有するモノマー溶液に対し、ＡＰＳ０．００２ｍｏｌ／ＬおよびＴＥＭＥＤ０．００６ｍｏｌ／Ｌとなるように添加混合後、分離層の上に注入して２５℃下で重合させ、電気泳動用ポリアクリルアミドゲルの濃縮層を得た。 Further, a monomer containing an acrylamide concentration of 5% by mass (acrylamide + crosslinking agent, total concentration), N, N-methylenebisacrylamide 3% by mass (based on acrylamide concentration), and a gel buffer having the composition described in Table 1. After adding and mixing the solution so as to be APS 0.002 mol / L and TEMED 0.006 mol / L, the solution was injected onto the separation layer and polymerized at 25 ° C. to obtain a concentrated layer of polyacrylamide gel for electrophoresis. .
泳動試験方法
トリス０．０２５ｍｏｌ／Ｌ、グリシン０．１９２ｍｏｌ／Ｌ、ＳＤＳ０．１質量％のレムリー処方の泳動緩衝液を用いて電気泳動を実施した。蛋白質試料としてαカゼイン、脱りん酸化αカゼインを電気泳動した。脱りん酸化αカゼインは、αカゼイン溶解液にアルカリフォスファターゼ（ニッポンジーン製）を加えた後１晩静置して脱りん酸化処理した。
 電気泳動は２０ｍＡ定電流で行い、泳動末端が下から１０ｍｍの所で通電を中止した。
  染色は、０．０５質量％クマシーブリリアントブルー(以下、ＣＢＢと略す)Ｇ－２５０、１２容量％酢酸、３０容量％メタノール溶液中で振とう機を使用して６０分間行い、脱色は１２容量％酢酸、１５容量％メタノール溶液中で振とう機を使用して１２０分間行った。
αカゼインおよび脱りん酸化αカゼインの移動度およびその差とそれぞれのバンドの鮮明さを表２－１に示す。
移動度とは蛋白質の移動距離をパーセントで表したものである。
（移動度％）＝(ウェル下端から各バンドの位置までの距離)
　　　　　　　　／(ウェル下端から泳動末端までの距離)×１００ Electrophoresis test method Electrophoresis was carried out 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, 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
実施例－１と同様の方法で作成したプレキャストゲルを冷蔵下で８ヶ月間保管した後、実施例－１と同様の方法で泳動試験を行った。結果を表２－１の実施例－２に示す。 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.
表１の実施例－３のゲル緩衝液を用いてプレキャストゲルを作成した以外は実施例－１と同様に泳動試験を実施した。結果を表２－１の実施例－３に示す。 An electrophoresis test was performed in the same manner as in Example 1 except that a precast gel was prepared using the gel buffer of Example 3 in Table 1. The results are shown in Example 3 in Table 2-1.
アクリルアミド濃度７．５％（アクリルアミド＋架橋剤、対全量濃度）に変更し、表１の実施例－４のゲル緩衝液を用いプレキャストゲルを作成した以外は実施例－1と同様に泳動試験を実施した。結果を表２－１の実施例－４に示す。 The migration test was performed in the same manner as in Example-1, except that the acrylamide concentration was changed to 7.5% (acrylamide + crosslinking agent vs. total concentration) and the precast gel was prepared using the gel buffer of Example-4 in Table 1. Carried out. The results are shown in Example 4 in Table 2-1.
表１の実施例－５のゲル緩衝液を用いた以外は実施例－１と同様の方法でプレキャストゲルを作成した。トリス０．１ｍｏｌ／Ｌ、トリシン０．１ｍｏｌ／Ｌ、ＳＤＳ０．１質量％のシャガー処方の泳動緩衝液を用いて電気泳動を実施した。蛋白質試料および検出方法は実施例１と同様にした。結果を表２－１の実施例－５に示す。 A precast gel was prepared in the same manner as in Example 1 except that the gel buffer of Example-5 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-5 in Table 2-1.
（比較例１）
分離層にｐＨ８．８の０．３７５ｍｏｌ／Ｌのトリス－塩酸ゲル緩衝液を、濃縮層にｐＨ６．８の０．１２５ｍｏｌ／Ｌトリス－塩酸ゲル緩衝液を用いてプレキャストゲルを作成した以外は実施例－1と同様に泳動試験を実施した。結果を表２－２の比較例－１に示す。 (Comparative Example 1)
Performed except that a precast gel was prepared using 0.375 mol / L Tris-hydrochloric acid gel buffer pH 8.8 for the separation layer and 0.125 mol / L Tris-hydrochloric acid gel buffer pH 6.8 for the concentration layer. A migration test was performed as in Example-1. The results are shown in Comparative Example-1 in Table 2-2.
（比較例－２）
比較例－１と同様の方法で作成したプレキャストゲルを冷蔵下で６ヶ月間保管した後、実施例－１と同様の方法で泳動試験を行った。結果を表２－２の比較例－２に示す。 (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.
（比較例－３）
分離層にｐＨ６．８の０．３７５ｍｏｌ／Ｌのトリス－塩酸ゲル緩衝液を、濃縮層にｐＨ６．８の０．１２５ｍｏｌ／Ｌトリス－塩酸ゲル緩衝液ゲル緩衝液を用いてプレキャストゲルを作成した以外は実施例－1と同様に泳動試験を実施した。結果を表２－２の比較例－３に示す。 (Comparative Example-3)
A precast gel was prepared using a 0.375 mol / L Tris-HCl gel buffer solution at pH 6.8 in the separation layer and a 0.125 mol / L Tris-HCl gel buffer gel buffer solution at pH 6.8 in the concentration layer. A migration test was conducted in the same manner as in Example-1 except for the above. The results are shown in Comparative Example-3 in Table 2-2.
（比較例－４）
前記化合物（Ｂ）を加えなかった以外は実施例－１と同様にプレキャストゲルを作成し泳動試験を実施した。結果を表２－２の比較例－４に示す。 (Comparative Example-4)
A precast gel was prepared and a migration test was conducted in the same manner as in Example 1 except that the compound (B) was not added. The results are shown in Comparative Example-4 in Table 2-2.
（表１） ゲル緩衝液組成
Figure JPOXMLDOC01-appb-I000006
ＭＯＰＳ：３－Ｎ－モルホリノプロパンスルホン酸
ＨＥＰＥＳ：Ｎ－２－ヒドロキシエチルピペラジン－Ｎ‘－２－エタンスルホン酸
  (Table 1) Gel buffer composition
Figure JPOXMLDOC01-appb-I000006
MOPS: 3-N-morpholinopropanesulfonic acid HEPES: N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
（表２－１）　移動度(%)
Figure JPOXMLDOC01-appb-I000007
  (Table 2-1) Mobility (%)
Figure JPOXMLDOC01-appb-I000007
（表２－２）　移動度(%)
Figure JPOXMLDOC01-appb-I000008
(Table 2-2) Mobility (%)
Figure JPOXMLDOC01-appb-I000008
表２－１に示すとおり実施例－１と３から５のプレキャストゲルでは、りん酸化αカゼインと脱りん酸化αカゼインはいずれも鮮明なバンドとなり明確な移動度差を得ることができ、本発明のプレキャストゲルはレムリーまたはシャガーの泳動用緩衝液を用いて蛋白質をそのりん酸化状態により分離することができた。 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.
表２－１に示すとおり実施例－２から、本発明の電気泳動用プレキャストゲルは作成後６ヶ月経過しても、作成直後と同じバンド鮮明さ、移動度、りん酸化状態による移動度差を得ることができた。 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 difference in band sharpness, mobility, and mobility due to phosphorylation state as those immediately after the production were obtained. I was able to get it.
表２－２に示すとおりレムリー処方のゲル緩衝液を用いた比較例－１と３のプレキャストゲルでは、作成直後は明確な移動度差を得ることができたが、りん酸化－脱りん酸化たんぱく質の移動度差は実施例－１に比べて小さく、また作成後６ヶ月経過後には、鮮明なバンドを得ることができなかった。 As shown in Table 2-2, in the precast gels of Comparative Examples 1 and 3 using the gel buffer of the Remley formulation, a clear mobility difference was obtained immediately after 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.
表２－２に示すとおり比較例－２のプレキャストゲルでは、明確な移動度差を得ることができなかった。バンドも不鮮明だった。 As shown in Table 2-2, a clear difference in mobility could not be obtained with the precast gel of Comparative Example-2. The band was also unclear.
表２－２に示すとおり比較例－４のプレキャストゲルでは、鮮明なバンドを得ることはできたが、αカゼインと脱りん酸化αカゼインの間に明確な移動度差を得ることができなかった。 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. .
以上の結果から、本発明の電気泳動用プレキャストゲルが、蛋白質のりん酸化状態を明確に分離することが可能であり、かつプレキャストゲルに必要とされる長期安定性を持つことは明らかである。 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.
本発明の電気泳動用プレキャストゲルを用いることにより、蛋白質の分離能及びゲルの形状が長期安定で、分析方法が普及しているレムリーあるいはシャガーの泳動緩衝液が使用でき使用者が経済的かつ効率的に、蛋白質のりん酸化修飾状態の分析をすることが可能となり、産業上の利用可能性は甚だ大きい。
 
 
 
  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 (8)

  1. その構造中の少なくとも一部に下記式（Ａ）で表される構造を有し、下記組成（１）および（２）の緩衝液を含有するアクリルアミド系共重合体水性ゲルであることを特徴とする電気泳動用プレキャストゲル。
    （１）トリス(ヒドロキシメチル)アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンと1種以上の両性電解質。
    （２）ｐＨが６．０～６．８。
    Figure JPOXMLDOC01-appb-C000001
      
    構造単位（Ａ）
    式中、Ｍ２+は遷移金属イオン　     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.
    (2) pH is 6.0 to 6.8.
    Figure JPOXMLDOC01-appb-C000001
    Structural unit (A)
    In the formula, M 2+ is a transition metal ion.
  2. ゲル緩衝液中の１種以上の両性電解質が、グリシンおよび／またはトリシンを必須とすることを特徴とする請求項１に記載の電気泳動用プレキャストゲル。 The precast gel for electrophoresis according to claim 1, wherein the one or more ampholytes in the gel buffer essentially contain glycine and / or tricine.
  3. １種以上の両性電解質において、グリシンおよび／またはトリシン以外の両性電解質の塩基解離定数の範囲が６．６～９．６であることを特徴とする請求項１あるいは２に記載の電気泳動用プレキャストゲル。 3. The precast for electrophoresis according to claim 1, wherein the base dissociation constant of the amphoteric electrolytes other than glycine and / or tricine is 6.6 to 9.6 in one or more ampholytes. gel.
  4. グリシンおよび／またはトリシン以外の両性電解質が、セリン、スレオニン、フェニルアラニン、グルタミン酸、トリプトファン、メチオニン、アラニン、バリン、アスパラギン酸、
    Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン、トリス（ヒドロキシメチル)メチルアミノプロパンスルホン酸、２－アミノエチルスルホン酸、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）－２－アミノエタンスルホン酸、３－Ｎ－モルホリノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジン－Ｎ‘－２－エタンスルホン酸、Ｎ－２－ヒドロキシエチルピペラジンプロパンスルホン酸、グリシルグリシン、トリス（ヒドロキシメチル）メチルアミノプロパンスルホン酸、Ｎ－トリス（ヒドロキシメチル）メチル－２－アミノエタンスルホン酸、２－アミノエチルスルホン酸から選択される一種以上であることを特徴とする請求項３に記載の電気泳動用プレキャストゲル。     Amphoteric electrolytes other than glycine and / or tricine are 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 It is at least one selected from sulfonic acid, glycylglycine, tris (hydroxymethyl) methylaminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, and 2-aminoethylsulfonic acid. Electrophoretic precast according to claim 3 Gel.
  5. グリシンおよび／またはトリシン以外の両性電解質が、グリシンおよび／またはトリシンに対して０．１～１００ｍｏｌ％の範囲で含まれることを特徴とする請求項１～４に記載の電気泳動用プレキャストゲル。 The precast gel for electrophoresis according to claim 1, wherein the ampholyte other than glycine and / or tricine is contained in an amount of 0.1 to 100 mol% with respect to glycine and / or tricine.
  6. ゲル中のトリス（ヒドロキシメチル）アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタンの濃度が、０．０７～０．２ｍｏｌ／Ｌであり、１種以上の両性電解質の濃度の合計が０．１～０．５ｍｏｌ／Ｌであることを特徴とする請求項１～５に記載の電気泳動用プレキャストゲル。 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 The precast gel for electrophoresis according to any one of Claims 1 to 5, wherein the total amount of is from 0.1 to 0.5 mol / L.
  7. アクリルアミド、下記化合物（Ｂ）、架橋剤、トリス(ヒドロキシメチル)アミノメタンおよび/またはビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン、および1種以上の両性電解質からなり、ｐＨが６．０～６．８である混合物水溶液を重合することを特徴とする電気泳動用プレキャストゲルの製造方法。
    Figure JPOXMLDOC01-appb-C000002
      
    化合物（Ｂ）
    式中、Ｍ２+は遷移金属イオン　     It consists of acrylamide, the following compound (B), a crosslinking agent, tris (hydroxymethyl) aminomethane and / or bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and one or more ampholytes, and has a pH of 6.0. A method for producing a precast gel for electrophoresis, comprising polymerizing an aqueous mixture solution of ˜6.8.
    Figure JPOXMLDOC01-appb-C000002
    Compound (B)
    In the formula, M 2+ is a transition metal ion.
  8. 請求項１～６に記載の電気泳動用プレキャストゲルと、ドデシル硫酸塩が存在するトリス(ヒドロキシメチル)アミノメタン及び両性電解質を含有する泳動用緩衝液を用い、蛋白質のりん酸化修飾状態を分析することを特徴とする電気泳動用プレキャストゲルの使用方法。
     
     
     
     
     
     
     
     
     
     
     
     
     
          Using the electrophoresis precast gel according to any one of claims 1 to 6 and an electrophoresis buffer containing tris (hydroxymethyl) aminomethane containing dodecyl sulfate and an amphoteric electrolyte, the phosphorylation modification state of the protein is analyzed. The use method of the precast gel for electrophoresis characterized by the above-mentioned.













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JP2001159621A (en) * 1999-12-02 2001-06-12 Hymo Corp Polyacrylamide precast gel for electrophoresis and method of manufacturing therefor and use of the same
WO2007015312A1 (en) * 2005-08-01 2007-02-08 Nard Institute, Ltd. Polyacrylamide gel for electrophoresis, polyacrylamide gel electrophoresis method using the same, method of producing the same and acrylamide compound

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JP2001159621A (en) * 1999-12-02 2001-06-12 Hymo Corp Polyacrylamide precast gel for electrophoresis and method of manufacturing therefor and use of the same
WO2007015312A1 (en) * 2005-08-01 2007-02-08 Nard Institute, Ltd. Polyacrylamide gel for electrophoresis, polyacrylamide gel electrophoresis method using the same, method of producing the same and acrylamide compound

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