WO2010064638A1 - 芳香族スルホン酸化合物を用いた新規Clear Native電気泳動法 - Google Patents
芳香族スルホン酸化合物を用いた新規Clear Native電気泳動法 Download PDFInfo
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- WO2010064638A1 WO2010064638A1 PCT/JP2009/070199 JP2009070199W WO2010064638A1 WO 2010064638 A1 WO2010064638 A1 WO 2010064638A1 JP 2009070199 W JP2009070199 W JP 2009070199W WO 2010064638 A1 WO2010064638 A1 WO 2010064638A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
- C07C309/01—Sulfonic acids
- C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
- C07C309/45—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton
- C07C309/46—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton having the sulfo groups bound to carbon atoms of non-condensed six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
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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
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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 mainly relates to an electrophoresis reagent, an electrophoresis composition, an electrophoresis kit, and a protein separation method.
- the present invention can perform electrophoretic separation of proteins in a negatively charged state as a whole while maintaining a higher-order structure and a complex structure, and can perform electrophoresis in a transparent state.
- a main object is to provide a reagent for electrophoresis of proteins, a composition for electrophoresis, a protein separation method using the same, and a kit for electrophoresis.
- the present inventor has found that a specific compound has almost no absorption in the wavelength region of visible light, and that the Blue-Native-electrophoresis method and The present inventors have found that it is possible to obtain almost the same electrophoresis pattern, and have further intensively studied to complete the present invention.
- Item 1 An electrophoresis reagent comprising at least one compound which can bind to a protein in a neutral electrophoresis buffer to form a negatively charged complex as a whole and is substantially colorless.
- Item 2. The electrophoresis reagent according to Item 1, wherein the compound has at least one arylsulfonic acid moiety.
- Item 3. Consisting of at least one compound that can bind to a protein in a neutral electrophoresis buffer to form a negatively charged complex of the whole molecule and is substantially colorless, Formula (I)
- R represents a hydrogen atom, SO 3 H or a salt thereof, a halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, Alkoxycarbonylamino, carbamoyl, mono- or dialkylcarbamoyl, sulfamoyl, mono- or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, carboxyl or NR 1 R 2 are shown.
- R ′ represents a hydrogen atom, SO 3 H or a salt thereof, a halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, alkoxycarbonylamino , Carbamoyl, mono- or dialkylcarbamoyl, sulfamoyl, mono- or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, carboxyl or NR 3 R 4 .
- R ′′ represents a hydrogen atom, SO 3 H or a salt thereof, a halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, alkoxycarbonylamino , Carbamoyl, mono- or dialkylcarbamoyl, sulfamoyl, mono- or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, carboxyl or NR 5 R 6 .
- R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are the same or different and each represents a hydrogen atom, alkyl, cycloalkyl, optionally substituted aryl, or optionally substituted aralkyl
- R a , R b , and R c are the same or different and SO 3 H or a salt thereof, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl , Cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, mono or dialkylcarbamoyl, sulfamoyl, mono or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbon
- Z represents a hydrogen atom, a halogen atom, OH, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, SH, alkylthio, alkyl, amino (NH 2 ), monoalkylamino, dialkylamino or acylamino.
- the compound of formula (I) has 1, 2, 3, 4, 5 or 6 SO 3 H or a salt thereof.
- Item 3. The electrophoresis reagent according to Item 1 or 2, represented by: Item 4.
- Item 4. The electrophoresis reagent according to any one of Items 1 to 3, comprising at least one compound represented by the following general formula (Ia):
- R ⁇ 1 >, R ⁇ 3 >, R ⁇ 5 > is the same or different and shows a hydrogen atom, alkyl, cycloalkyl, the aryl which may be substituted, or the aralkyl which may be substituted.
- R a , R b , R c , R 2a , R 4a , R 6a are the same or different, and SO 3 H or a salt thereof, a halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoro Methoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, mono or dialkylcarbamoyl, sulfamoyl, mono or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, Alkylcarbonyloxy, arylcarbonyloxy, aryloxy, acylamino or carboxyl is shown.
- N1, n2, and n3 are the same or different and represent an integer of 0 to 4.
- M1, m2, and m3 are the same or different and represent an integer of 0 to 5.
- the compound of formula (Ia) has 1, 2, 3, 4, 5 or 6 SO 3 H or a salt thereof, and R 1 , R 3 , R 5 ,
- Item 5 The electrophoresis reagent according to any one of Items 1 to 4, comprising at least one compound represented by the following formula (Ib):
- R 1 , R 3 and R 5 are the same or different and each represents a hydrogen atom, C 1-4 alkyl, an optionally substituted phenyl or an optionally substituted benzyl, R a and R c are the same or different and are a hydrogen atom or methyl.
- R 2b and R 2c represents a hydrogen atom, and the other represents SO 2 NR 9 R 10 , SO 3 H or a salt thereof.
- R 4b and R 4c are a hydrogen atom, and the other is alkoxy.
- R 6b and R 6c is a hydrogen atom, and the other is SO 2 NR 9 R 10 , SO 3 H or a salt thereof.
- R 9 and R 10 represent hydrogen or alkyl.
- R a and R c are the same or different and are a hydrogen atom or methyl.
- R 2b and R 6b represent SO 3 H or a salt thereof.
- Item 7. An electrophoresis gel composition comprising an electrophoresis gel and the electrophoresis reagent according to any one of Items 1 to 6.
- Item 8. An electrophoresis buffer composition comprising an electrophoresis buffer and the electrophoresis reagent according to any one of Items 1 to 6.
- Item 9. Item 7. A method for separating a protein, wherein the protein is subjected to Clear Native electrophoresis by adding the electrophoresis reagent according to any one of Items 1 to 6 to a protein sample and / or an electrophoresis buffer.
- Item 10. Item 9.
- a protein electrophoresis kit characterized by Item 11.
- M is a hydrogen atom or a metal forming a water-soluble salt or N (R 8 ) 4 (R 8 is the same or different and is a hydrogen atom, alkyl, alkoxyalkyl, hydroxyalkyl, optionally substituted aryl) Or an optionally substituted aralkyl.)
- electrophoresis can be performed in a transparent state, and a negative charge is generally obtained while maintaining a higher-order structure or a complex structure. Protein separation can be performed.
- the electrophoresis reagent of the present invention is substantially colorless, it is not necessary to decolorize after electrophoresis, and proteins can be detected with high sensitivity by direct silver staining or the like. Blue-Native electrophoresis It has features that are much more sensitive than the method.
- the measurement result of the visible absorption spectrum of compound (Ig) and CBB G250 is shown.
- the vertical axis represents absorbance and the horizontal axis represents wavelength (unit: nm).
- the solid line in FIG. 1 shows the measurement result of the 0.0167 mg / ml CBB G250 solution.
- the dotted line in FIG. 1 shows the measurement results of a 1 mg / ml compound (Ig) solution.
- BN-PAGE Blue Native PAGE
- CN-PAGE Clear Native PAGE
- the left side of FIG. 2 shows a pattern in BN-PAGE.
- FIG. 2 shows the pattern in CN-PAGE.
- the numbers on the horizontal axis in FIG. 2 are lane numbers assigned according to the type of protein, 1: thyroglobulin (669 kDa), 2: ferritin (440 kDa), 3: aldolase (158 kDa), 4: Albumin (Conalbumin: 75kDa), 5: Ovalbumin (43kDa), 6: Carbonic anhydrase (29kDa), 7: Ribonuclease A (RNase A: ⁇ ⁇ 13.7kDa), 8: Nitric oxide reductase (NOR: 68.5 kDa), 9: Band 3 (Band3: 55 kDa (110 kDa))), 10: Adenosine receptor A2a (A2a: 33 kDa).
- FIG. 3 shows the result of fluorescence imaging of the gel after CN-PAGE.
- the right side of FIG. 3 shows the result of detection by CBB staining of the gel after CN-PAGE.
- Lane 1 shows the marker
- Lane 2 shows EGFP
- Lane 3 shows the membrane protein-EGFP fusion expression membrane fraction solubilized with dodecyl maltoside (DDM).
- DDM dodecyl maltoside
- FIG. 4 shows the results of BN-PAGE.
- the right side of FIG. 4 shows the results of CN-PAGE.
- FIG. 5 (A) shows the results of the NOR sample.
- FIG. 5 (B) shows the result of the BSA sample.
- the vertical axis in FIG. 5 indicates the molecular weight (kDa).
- the horizontal axis in FIG. 5 indicates the lane number assigned according to the amount of the electrophoresed protein.
- M marker, 1: 1000 ng, 2: 500 ng, 3: 100 ng, 4: 50 ng, 5: 10 ng, 6: 5 ng, 7: 1 ng, 8: 0.5 ng, 9: 0.1 ng.
- the electrophoresis reagent used in the present invention contains a substantially colorless compound that forms an anion in an aqueous neutral solution with an aryl moiety that binds to the hydrophobic portion of the protein. SO 3 H or a salt thereof. When the compound coexists with protein in an aqueous solution, an anionic complex is formed as a whole.
- the compound contained in the electrophoresis reagent has at least one aryl sulfonate moiety.
- the compound can further have at least one arylamino moiety.
- R d and R e are the same or different, SO 3 H or a salt thereof, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl) , Cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, mono or dialkylcarbamoyl, sulfamoyl, mono or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryl Represents oxy, acylamino or carboxyl.
- R 7 represents a hydrogen atom, alkyl, cycloalkyl, aryl which may be substituted or aralkyl which may be substituted.
- N4 is an integer from 0 to 4.
- n5 represents an integer of 0 to 5.
- M is a hydrogen atom or a metal that forms a water-soluble salt, particularly an alkali metal such as Na, K, Li, or Cs, or N (R 8 ) 4 (R 8 is the same or different and is a hydrogen atom, alkyl, alkoxyalkyl, A quaternary ammonium represented by hydroxyalkyl, an optionally substituted aryl or an optionally substituted aralkyl. )
- aryl sulfonic acid moiety (aryl sulfonate moiety), aryl aryl moiety (aryl amino moiety), where aryl is naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl, chromanyl
- arylsulfonic acid moieties (aryl sulfonate moiety)
- arylamino moieties aryl amino moiety
- 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl, fluorenyl and phenanthryl are also included. It is included in the present invention.
- Methylenedioxy (—O—CH 2 —O—) is formed by combining two adjacent substituents together.
- the compound is represented by formula (I).
- R, R ′, R ′′, R a , R b , R c , n1, n2, n3 and Z are as defined above.
- the compound is represented by formula (I ').
- R, R 3 , R 4 , R 5 , R 6 , R a , R b , R c , n1, n2, n3 and Z are as defined above.
- halogen atom means fluorine, chlorine, bromine and iodine, and fluorine, chlorine and bromine are preferable.
- Alkyl includes, for example, straight-chain, branched, Examples thereof include branched or cyclic C 1-10 alkyl, preferably C 1-6 alkyl, and more preferably C 1-4 alkyl. Particularly preferred alkyl is methyl or ethyl.
- Cycloalkyl includes C 3-7 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
- Haldroxyalkyl includes, for example, C 1-4 alkyl having one OH such as hydroxymethyl, hydroxyethyl, hydroxy n-propyl, hydroxyisopropyl, hydroxy n-butyl, hydroxyisobutyl, hydroxytert-butyl and the like. .
- Alkoxyalkyl includes, for example, C 1-4 alkoxy C 1-4 alkyl such as methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl and the like.
- Aryl means a monocyclic or polycyclic system consisting of a 5- or 6-membered aromatic hydrocarbon ring. Specific examples include phenyl, naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl, chromanyl, 2, Examples include 3-dihydro-1,4-dioxanaphthalenyl, indanyl, fluorenyl and phenanthryl.
- Alkyl means straight, branched or cyclic C 1-4 alkyl substituted with aryl as described above, specifically benzyl, 1-phenylethyl, 2-phenylethyl, 1 -Phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, naphthylmethyl and the like.
- Alkenyl means one having at least one double bond, such as vinyl, allyl, 1-propenyl, 2-methyl-2-propenyl, isopropenyl, 1-, 2- or 3-butenyl, 2 -, 3- or 4-pentenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 5-hexenyl, 1-cyclopentenyl, 1-cyclohexenyl, 3-methyl-3-butenyl, etc. , Branched or cyclic C 2-10 alkenyl, preferably C 2-6 alkenyl, more preferably C 2-4 alkenyl.
- Alkynyl means one having at least one triple bond, for example, straight chain such as ethynyl, 1- or 2-propynyl, 1-, 2- or 3-butynyl, 1-methyl-2-propynyl, etc. Branched or cyclic C 2-10 alkynyl, preferably C 2-6 alkynyl, more preferably C 2-4 alkynyl.
- Alkoxy includes, for example, linear, branched or cyclic C 1- 1 such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy and hexyloxy. 6 alkoxy is mentioned.
- Examples of “monoalkylamino” include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino, n-pentylamino, isopentylamino, hexylamino and the like. Examples include linear, branched, or cyclic mono-C 1-6 alkylamino.
- dialkylamino examples include dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, ditert-butylamino, di-n-pentylamino, diisopentylamino, dihexylamino And straight chain, branched chain or cyclic di-C 1-6 alkylamino.
- Alkoxycarbonylamino includes methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, isopropoxycarbonylamino, butoxycarbonylamino, isobutoxycarbonylamino, tert-butoxycarbonylamino, pentyloxycarbonylamino, isopentyloxycarbonylamino And linear, branched or cyclic C 1-6 alkoxycarbonylamino such as hexyloxycarbonylamino.
- “Monoalkylcarbamoyl” includes C 1 such as methylcarbamoyl, ethylcarbamoyl, n-propylcarbamoyl, isopropylcarbamoyl, n-butylcarbamoyl, isobutylcarbamoyl, tert-butylcarbamoyl, n-pentylcarbamoyl, isopentylcarbamoyl, hexylcarbamoyl and the like. -6 alkyl monocarbamoyl is mentioned.
- Dialkylcarbamoyl includes dimethylcarbamoyl, diethylcarbamoyl, di-n-propylcarbamoyl, diisopropylcarbamoyl, din-butylcarbamoyl, diisobutylcarbamoyl, ditert-butylcarbamoyl, din-pentylcarbamoyl, diisopentylcarbamoyl, dihexylcarbamoyl And di-C 1-6 alkylcarbamoyl.
- “Monoalkylsulfamoyl” includes methylsulfamoyl, ethylsulfamoyl, n-propylsulfamoyl, isopropylsulfamoyl, n-butylsulfamoyl, isobutylsulfamoyl, tert-butylsulfamoyl And C 1-6 alkyl monosulfamoyl such as n-pentylsulfamoyl, isopentylsulfamoyl, hexylsulfamoyl and the like.
- Dialkylsulfamoyl includes dimethylsulfamoyl, diethylsulfamoyl, di-n-propylsulfamoyl, diisopropylsulfamoyl, di-n-butylsulfamoyl, diisobutylsulfamoyl, ditert-butylsulfone Examples include diC 1-6 alkylsulfamoyl such as famoyl, di-n-pentylsulfamoyl, diisopentylsulfamoyl, dihexylsulfamoyl and the like.
- Alkylsulfonylamino includes methylsulfonylamino, ethylsulfonylamino, n-propylsulfonylamino, isopropylsulfonylamino, n-butylsulfonylamino, isobutylsulfonylamino, tert-butylsulfonylamino, n-pentylsulfonylamino, isopentyl Examples thereof include linear, branched or cyclic C 1-6 alkylsulfonylamino such as sulfonylamino and hexylsulfonylamino.
- Alkoxycarbonyl '' includes straight chain such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl and hexyloxycarbonyl, Examples thereof include branched or cyclic C 1-6 alkoxycarbonyl.
- “Monoarylamino” includes phenylamino, naphthylamino, fluorenylamino, anthrylamino, biphenylylamino, tetrahydronaphthylamino, chromanamino, 2,3-dihydro-1,4-dioxanaphthalenyl Amino, indanylamino, fluorenylamino and phenanthrylamino are mentioned.
- Diarylamino includes diphenylamino, dinaphthylamino, difluorenylamino, dianthrylamino, dibiphenylylamino, ditetrahydronaphthylamino, dichromanylamino, di (2,3-dihydro-1,4-dio Xanaphthalenyl) amino, diindanylamino, difluorenylamino and diphenanthrylamino.
- “Monoaralkylamino” includes benzylamino, 1-phenylethylamino, 2-phenylethylamino, 1-phenylpropylamino, 2-phenylpropylamino, 3-phenylpropylamino, naphthylmethylamino.
- Diaralkylamino includes dibenzylamino, diphenethylamino, dipropylamino, dinaphthylmethylamino.
- Alkylcarbonyloxy includes methylcarbonyloxy, ethylcarbonyloxy, n-propylcarbonyloxy, isopropylcarbonyloxy, n-butylcarbonyloxy, isobutylcarbonyloxy, tert-butylcarbonyloxy, n-pentylcarbonyloxy, isopentyl Examples thereof include linear, branched or cyclic C 1-6 alkylcarbonyloxy such as carbonyloxy and hexylcarbonyloxy.
- Arylcarbonyloxy includes phenylcarbonyloxy, naphthylcarbonyloxy, fluorenylcarbonyloxy, anthrylcarbonyloxy, biphenylylcarbonyloxy, tetrahydronaphthylcarbonyloxy, chromanylcarbonyloxy, 2,3-dihydro-1, 4-Dioxanaphthalenylcarbonyloxy, indanylcarbonyloxy, fluorenylcarbonyloxy and phenanthrylcarbonyloxy are mentioned.
- Aryloxy includes phenyloxy, naphthyloxy, fluorenyloxy, anthryloxy, biphenylyloxy, tetrahydronaphthyloxy, chromanyloxy, 2,3-dihydro-1,4-dioxanaphthalenyloxy , Indanyloxy, fluorenyloxy and phenanthryloxy.
- Acylamino includes arylcarbonylamino such as C 1-6 alkanoylamino such as formylamino, acetylamino, propionylamino, butyrylamino and benzoylamino.
- alkylcarbonyloxy examples include methylcarbonyloxy, ethylcarbonyloxy, n-propylcarbonyloxy, isopropylcarbonyloxy, n-butylcarbonyloxy, isobutylcarbonyloxy, tert-butylcarbonyloxy, n-pentylcarbonyloxy, iso Examples thereof include C 1-6 alkylcarbonyloxy such as pentylcarbonyloxy and hexylcarbonyloxy.
- arylcarbonyloxy examples include phenylcarbonyloxy, naphthylcarbonyloxy, fluorenylcarbonyloxy, anthrylcarbonyloxy, biphenylylcarbonyloxy, tetrahydronaphthylcarbonyloxy, chromanylcarbonyloxy, 2,3-dihydro-1 , 4-dioxanaphthalenylcarbonyloxy, indanylcarbonyloxy, fluorenylcarbonyloxy and phenanthrylcarbonyloxy.
- alkylthio examples include linear, branched or cyclic C 1 such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio, hexylthio and the like. -6 alkylthio is mentioned.
- substituents for the optionally substituted aryl or the optionally substituted aralkyl include a halogen atom, alkyl, alkoxy, SO 3 H or a salt thereof, hydroxy, trifluoromethoxy, trifluoroethoxy, cyano, nitro, amino , Monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, mono or dialkylcarbamoyl, sulfamoyl, mono or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, acylamino or carboxyl And may have 1 to 4, preferably 1 to 3, more preferably 1 or 2 of these substituents.
- N1, n2, n3, m1, m2, and m3 are the same or different and each represents an integer of 0 to 4, preferably 0 to 3, more preferably 0, 1, or 2.
- the H salt is not particularly limited as long as it is water-soluble salts of SO 3 H, for example, SO 3 M (M is a metal which forms a hydrogen atom or a water-soluble salt, especially Na, K, Li, Cs Or an alkali metal such as N (R 8 ) 4 (R 8 is the same or different and represents a hydrogen atom, alkyl, alkoxyalkyl, hydroxyalkyl, optionally substituted aryl, or optionally substituted aralkyl.)
- M is a metal which forms a hydrogen atom or a water-soluble salt, especially Na, K, Li, Cs Or an alkali metal such as N (R 8 ) 4 (R 8 is the same or different and represents a hydrogen atom, alkyl, alkoxyalkyl, hydroxyalkyl, optionally substituted aryl, or optionally substituted aralkyl.
- a sulfonate salt represented by the following formula:
- the compound of the present invention has at least one SO 3 H (sulfo group) or a salt thereof as a substituent. This is because the compound of the present invention binds to a protein through a sulfo group and electrophoreses with a negative overall charge. Dyes such as CBB G250 and CBB R250 used in conventional Blue-Native electrophoresis have two sulfo groups, but have an ammonium cation in the molecule, and thus have a monovalent negative charge as a whole molecule. The compound of the present invention has at least a monovalent negative charge as a whole molecule.
- the number of SO 3 H or a salt thereof (1 to 6) may be two or more, but one is sufficient.
- the compounds (Ig) and (Ih) of the present invention produced by reducing CBB G250 and CBB R250 have two SO 3 H or a salt thereof and exist as a divalent anion in an aqueous solution.
- the dissociation of subunits and the partial destruction of tertiary structure may occur depending on the type of protein or electrophoresis conditions.
- sulfo group to a functional group having no charge, such as a hydrogen atom, a hydroxyl group (OH), sulfamoyl (—SO 2 NH 2 ), mono- or dialkylsulfamoyl, etc.
- a functional group having no charge such as a hydrogen atom, a hydroxyl group (OH), sulfamoyl (—SO 2 NH 2 ), mono- or dialkylsulfamoyl, etc.
- the negative charge is monovalent.
- the structure of the protein may be maintained by mildly acting on the protein.
- Preferred compounds of the present invention include compounds (Ii), (Ij), (Ik) and (Il) in addition to (Ig) and (Ih).
- R 9 and R 10 are the same or different and each represents a hydrogen atom or alkyl.
- one sulfo group may be substituted with a sulfonamide or the like, which has no charge and is water-soluble.
- substituting with another group having polarity it is possible to lead to a colorless compound having the same charge as the dye compound while retaining the same protein binding property and water solubility as the original dye compound. .
- R, R ′, R ′′, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , R c , n1, n2, n3 and Z are as defined above. As defined.
- Compound (I) of the present invention can be synthesized in one step by reacting aldehyde (1) with compound (2) and compound (3) as shown in Scheme 1 (a1).
- the reaction uses about 1 mole of each of compound (2) and compound (3) and 1 to 4 moles of sulfuric acid in an amount from a catalytic amount to 1 to 1 mole of compound (1). It proceeds advantageously by reacting for a time.
- Compound (2) and compound (3) are preferably the same.
- the compound obtained by reacting two molecules of the compound (2) with the aldehyde (1), the compound obtained by reacting two molecules of the compound (3), the compound (2) and the compound (3) ) Can be produced one molecule at a time, and these can be separated by ordinary separation means such as column chromatography, recrystallization, solvent extraction, preparative HPLC, etc., to obtain each compound of formula (I).
- the solvent include alcohols such as methanol, ethanol and propanol, ethers such as tetrahydrofuran, dioxane, 1,2-dimethoxyethane and diglyme.
- This compound can be represented by the formula (1) by introducing Z other than a hydrogen atom by 1) Halogenation and, if necessary, 2) Substitution of a halogen atom to another functional group.
- the compound of I) can be obtained by halogenation by reacting with halogen molecules such as F 2 , Cl 2 , Br 2 , I 2, and halogenating agents such as N-bromosuccinimide and N-chlorosuccinimide.
- alkali metal hydroxide such as sodium hydroxide, (alkyl) -OH, (alkyl) -COOH, (aryl
- Compound (Id) of the present invention can be prepared by reacting aldehyde (1b) with compound (2b) and compound (3b) in the presence of a solvent and sulfuric acid as shown in Scheme 1 (b). It can be synthesized in stages. The reaction uses about 1 mole of each of compound (2b) and compound (3b) and 1 to 4 moles of sulfuric acid in an amount from a catalytic amount to 1 to 1 mole of aldehyde (1b). It proceeds advantageously by reacting for a time.
- compound (Id) can be led to sulfonic acid compound (Ie) by reacting at a temperature of room temperature to about 40 ° C. for 1 to 48 hours in the presence of a sulfonating agent such as concentrated sulfuric acid or fuming sulfuric acid. .
- a sulfonating agent such as concentrated sulfuric acid or fuming sulfuric acid.
- the compound (Ie) is oxidized with MnO 2 in the presence of acetic acid and HCl to obtain a compound (IIa), which is reacted with HNR 1 R 2 to obtain a compound of the formula (IIb).
- Compound (If) can be obtained by further reducing compound (IIb) with a reducing agent such as NaCNBH 3 .
- Compound (If) can be converted to compound (If ′) by introducing a Z group in the same manner as described above.
- Compound (IIa) can be obtained by reacting compound (Ie) with an equivalent to excess amount of MnO 2 at a temperature of about room temperature for 1 to 24 hours using acetic acid and HCl as a solvent.
- Compound (IIb) can be obtained by reacting 1 mol of compound (IIa) with 1 mol to an excess amount of HNR 1 R 2 in a solvent such as alcohol such as methanol and chlorinated hydrocarbon such as methylene chloride.
- Compound (If) can be obtained by reducing the compound (IIb) with 1 reducing agent such as NaCNBH 3 in 1 equivalent to an excess amount in an alcohol such as methanol with respect to 1 mol of compound (IIb).
- Ar represents the same or different aryl group which may be substituted.
- R 5 and R 6 are as defined above.
- R f may be the same or different from SO 3 H or its Salt, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, Mono or dialkylcarbamoyl, sulfamoyl, mono or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, acylamino or carboxyl .N4 is 0-4 .Ar represents an integer, R 5, R 6, R f preferably has a whole 1-6 SO
- R a , R b , n1, n2 are as defined above.
- R f , R g represent an aryl sulfonic acid in which one is a hydrogen atom and the other is optionally substituted?
- R f and R g together represent an aromatic or heteroaromatic group having a sulfo group.
- optionally substituted aryl sulfonic acid examples include an optionally substituted benzene sulfonic acid, an optionally substituted naphthalene sulfonic acid, an optionally substituted anthracene sulfonic acid, and an optionally substituted phenanthrene sulfone.
- Acid optionally substituted fluorene sulfonic acid, optionally substituted indane sulfonic acid, optionally substituted indene sulfonic acid, optionally substituted tetralin sulfonic acid, optionally substituted acenaphthene A sulfonic acid is mentioned.
- Examples of the optionally substituted aromatic group having a sulfo group include those having a sulfo group (indene, indane, tetralin, fluorene).
- Examples of the optionally substituted heteroaromatic group having a sulfo group include those having a sulfo group (benzofuran, benzopyran, xanthene, chromene, isobenzofuranone, indoline, isoindoline). Specific compounds are shown below.
- R h and R i are the same or different and each represents a sulfo group (SO 3 H) or a salt thereof, a halogen atom, Alkyl, alkenyl, alkynyl, alkoxy, methylenedioxy, hydroxy, trifluoromethoxy, trifluoroethoxy, trifluoromethyl, cyano, nitro, amino, monoalkylamino, dialkylamino, alkoxycarbonylamino, carbamoyl, mono or dialkylcarbamoyl , Sulfamoyl, mono- or dialkylsulfamoyl, alkylsulfonylamino, alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aryloxy, acylamino or carboxyl, n5 and n6 each represent 0 to 4 Indicates
- CBB R150, CBB G250, CBB R250 and CBB R350 are preferable.
- the compounds represented by the formulas (Ig) and (Ih) can be obtained by reducing commercially available CBB G250 or CBB R250 according to a conventional method.
- the compound represented by the formula (Ia) can be easily obtained by synthesizing CBB compound (IIc) by the same synthesis method as CBB G250 or CBB R250 and reducing it with a reducing agent (Scheme). 2).
- R 1 , R 3 , R 5 , R a , R b , R c , R 2a , R 4a , R 6a , n1, n2, n3, m1, m2, m3 are as defined above. .
- the reduction can be performed in a solvent in the presence of a reducing agent such as sodium cyanoborohydride, sodium borohydride, LiAlH 4 .
- a reducing agent such as sodium cyanoborohydride, sodium borohydride, LiAlH 4 .
- the solvent include alcohols such as methanol and ethanol.
- the reduction reaction proceeds advantageously by using a reducing agent in an excess amount of 1 mole to 1 mole of the compound of the formula (IIc) and reacting at a temperature from 0 ° C. to the boiling point of the solvent for 30 minutes to 24 hours.
- M represents a hydrogen atom or a water-soluble salt.
- the compound (I) of the present invention has a protein binding ability.
- Compound (I) has almost no visible light absorption and is almost colorless. Because of these properties, proteins such as proteins having a basic isoelectric point and membrane proteins can be given a negative charge without adding visible light absorption.
- Compound (I) has high solubility in water and is easy to handle.
- surfactants such as SDS, which have a strong protein-denaturing action, they bind relatively weakly to the molecular surface of the protein, and therefore do not change the higher-order protein structure or the association state of multimeric proteins.
- Compound (I) can be used as an electrophoresis reagent (Clear Native electrophoresis reagent) that allows electrophoresis of proteins in their natural state and makes the gel after electrophoresis colorless and transparent. Can be used.
- the electrophoresis reagent of the present invention may comprise at least one compound (I), an electrophoresis gel composition in which the compound (I) is blended in an electrophoresis gel, and an electrophoresis buffer (cathode buffer).
- An anode buffer), a gel preparation buffer, a sample buffer, and the like may be a buffer composition for electrophoresis in which compound (I) is blended.
- the gel for electrophoresis examples include polyacrylamide gel, agarose gel, dextran gel and the like.
- the present invention is suitably used for polyacrylamide gel electrophoresis in which the electrophoresis gel is a polyacrylamide gel.
- the gel may be a constant concentration gel or a concentration gradient gel.
- concentration gradient gel has a sharper band than the constant concentration gel.
- the electrophoresis buffer (sample buffer), the same one as the sample buffer in the known Blue-Native electrophoresis can be used.
- the pH is about 6 to 8, and the salt concentration is about 0.3M or less. Things are used.
- 100 mM imidazole pH 7.0, 100 mM NaCl, 1% dodecyl maltoside, 20% glycerol, 1% compound (I), or 20 mM Hepes pH 7.5, 150 mM NaCl, 0.1% dodecyl maltoside, 20% Glycerol, 0.1% compound (I) composition or the like can be used.
- the buffers used for known Blue-Native electrophoresis can be used as they are for the electrophoresis buffer (cathode buffer, anode buffer) and gel preparation buffer.
- the cathode buffer includes 50 mM Tricine, 7.5 mM imidazole (pH 7.0), 0.02% compound (I), and the anode buffer includes 25 mM imidazole (pH 7.0).
- reagent of the present invention can be added to the reagent of the present invention depending on the purpose, such as improvement or stabilization of protein solubility.
- solubilizers examples include solubilizers and disulfide bond reducing reagents.
- solubilizer examples include n-dodecyl- ⁇ -D-maltoside (DDM), digitonin and the like. These solubilizers can increase the solubility without inhibiting the binding between compound (I) and protein.
- DDM n-dodecyl- ⁇ -D-maltoside
- disulfide bond reducing reagent examples include dithiothreitol and 2-mercaptoethanol.
- the protein separation method of the present invention is a method for separating proteins by electrophoresis using the above-described electrophoresis reagent or electrophoresis composition.
- the compound (I) contained in the electrophoresis reagent or composition of the present invention binds to the protein without destroying the higher-order structure of the complex or protein, negatively charges the whole molecule, and the protein has a natural structure. In this state, the protein can be separated by moving in the anode direction.
- the target protein sample is not particularly limited as long as it can be analyzed.
- protein types include enzymes, membrane proteins, and antibodies. It may be a protein that forms a complex.
- the molecular weight of the protein is not particularly limited as long as it can be analyzed, but is usually about 20,000 to 1,500,000 daltons, particularly about 50,000 to 1,000,000 daltons.
- the sample may be a protein purification solution, a cell disruption solution, a cell membrane solubilization solution, or the like.
- the sample may include one type of protein, or may include two or more types of protein.
- a solution containing two or more kinds of proteins considered to interact can be used as a sample.
- the sample may be pretreated before electrophoresis.
- pretreatment include an operation for removing large aggregates by centrifugation and filtration, and a protein fluorescent labeling treatment such as SYPRO®Orange.
- the above sample is prepared as an electrophoresis sample using the electrophoresis reagent or composition of the present invention.
- the sample can be prepared according to a known method.
- the protein sample may be mixed with a buffer composition containing compound (I) and a sample buffer to prepare a sample.
- a sample may be prepared by adding an electrophoresis reagent comprising Compound (I) to a buffer in which a protein sample is solubilized, and further adding other components such as glycerol as necessary.
- the protein concentration in the sample differs depending on the staining method and detection method after electrophoresis and cannot be uniquely set. However, when detecting by CBB staining, it is usually about 0.5 to 20 ⁇ g / well, especially about 1 to 10 ⁇ g / well. . When a fluorescent protein fusion is prepared and the fluorescence of the protein is detected, it is usually 0.05 ⁇ g to 1 ⁇ g / well, particularly about 0.1 to 0.5 ⁇ g / well. In addition, when detecting by silver staining, it is usually 0.5 to 100 ng / well, particularly about 5 to 50 ng / well. These concentrations are expressed with a capacity per well of about 20 ⁇ L.
- the concentration of compound (I) in the sample is also appropriately set according to the detection method and the like, but is usually about 0.02 to 0.5%, particularly about 0.05 to 0.25%.
- Proteins can be separated by applying the electrophoresis sample prepared as described above to a gel using a general-purpose electrophoresis apparatus and performing electrophoresis.
- electrophoresis apparatus those known for protein electrophoresis can be used as appropriate, and include, for example, an electrophoresis tank, a glass plate for electrophoresis, a buffer tank, a spacer, a comb, a clip, a power supply, a peristaltic pump, and the like. Common ones can be used.
- electrophoresis buffer the same buffer as that used in the known Blue-Native electrophoresis can be used.
- a Tris-glycine buffer an imidazole-tricine buffer, a bistris-tricine buffer, or the like can be used.
- Different compositions are used on the anode side and the cathode side.
- Tris-glycine buffer Cathode buffer: 25 mM Tris base, 192 mM glycine, pH 8.5 Anode buffer: 25 mM Tris base, 192 mM glycine, pH 8.5 The composition of this is mentioned.
- imidazole-tricine buffer Cathode buffer: 7.5 mM imidazole, 50 mM Tricine, pH 7.0 Anode buffer: 25 mM imidazole, pH 7.0 The composition of this is mentioned.
- Bistris-Tricine buffer Cathode buffer: 50 mM Bis-Tris, 50 mM Tricine, pH 6.8 Anode buffer: 50 mM Bis-Tris, 50 mM Tricine, pH 6.8 The composition of this is mentioned.
- compound (I) is usually contained in an amount of about 0.002 to 0.02%, particularly about 0.004 to 0.01%.
- Electrophoretic conditions other than those described above are not particularly limited as long as the protein can be electrophoresed while maintaining its natural state, and can be appropriately set according to the type of sample, the purpose of analysis, and the like.
- the temperature can also be set as appropriate according to the type of protein, etc. However, generally, proteins are more stable at low temperatures, so electrophoresis is usually performed at about 4 ° C.
- the protein in the sample is drawn to the anode while maintaining the natural state, and the protein is separated according to the size of the molecule.
- the protein band can be immobilized and visualized to analyze the size, purity, interaction, etc. of the protein in its natural state.
- the Clear® Native electrophoresis of the present invention is characterized by extremely high sensitivity.
- the gel after electrophoresis using the compound (I) is transparent, it is possible to detect color development or fluorescence derived from protein immediately after electrophoresis.
- a fusion protein of a fluorescent protein such as GFP and a protein to be studied is expressed in an appropriate expression host, and a cell disruption crude extract containing the chimeric fluorescent protein is electrophoresed and expressed, and the amount is associated without purification. You can check the status.
- enzyme activity measurement with color development can be performed as it is. Examples of enzyme activity measurement with color development include ATP hydrolysis reaction, peroxidase reaction, galactosidase reaction, dehydrogenase reaction and the like.
- electrophoresis combining the electrophoresis method using the compound (I) of the present invention with other electrophoretic separation such as SDS-PAGE is also possible.
- electrophoresis is performed in a state where the compound (I) of the present invention is formed to form a complex
- electrophoresis is performed in a dissociated state by performing SDS-PAGE.
- SDS-PAGE can be analyzed for higher order structures and polypeptide chains.
- electrophoresis reagent and composition of the present invention can be combined with various reagents and electrophoresis apparatus as necessary to form an electrophoresis kit.
- the electrophoresis buffer composition in addition to the electrophoresis reagent, the electrophoresis buffer composition, and the electrophoresis gel composition of the present invention, one or two kinds such as a molecular weight marker, a color reagent, a blotting buffer, and a blotting membrane are used.
- a molecular weight marker for example, a molecular weight marker, a color reagent, a blotting buffer, and a blotting membrane are used. The above can be included.
- the kit is provided with one or more general electrophoresis apparatuses or parts such as an electrophoresis tank, an electrophoresis glass plate, a buffer tank, a spacer, a comb, a clip, a power supply, or a peristaltic pump. You can also.
- one or more general electrophoresis apparatuses or parts such as an electrophoresis tank, an electrophoresis glass plate, a buffer tank, a spacer, a comb, a clip, a power supply, or a peristaltic pump. You can also.
- electrophoresis reagent and composition and the protein separation method and electrophoresis kit of the present invention can be added with known techniques relating to protein electrophoresis, particularly Native electrophoresis, as necessary. .
- the gel after electrophoresis is colorless and transparent, detection by silver staining or Western blotting is possible without washing the colored reagent contained in the gel.
- the gel immediately after electrophoresis is transparent, it is possible to detect color development and fluorescence derived from proteins as they are. Thereby, even a very small amount of protein in a crude sample can be analyzed with high accuracy. For example, by using the target protein as a fusion with GFP and observing the fluorescence of GFP, the association state and molecular weight can be analyzed with high accuracy immediately after electrophoresis. Furthermore, the enzyme activity of the protein electrophoresed while maintaining the activity can also be measured by a color reaction immediately after the electrophoresis.
- the cathode buffer can be made colorless and transparent, it can be easily confirmed whether the protein sample is properly loaded into the sample well, and the sample handling and the electrophoresis operation are also simplified.
- the present invention provides a method for simply and highly accurately analyzing a protein in a natural state or in a state in which an enzyme activity is maintained. This greatly contributes to the analysis of molecular complexes and the development of applied technologies.
- Blue Native PAGE is abbreviated as “BN-PAGE” and Clear Native PAGE is abbreviated as “CN-PAGE”.
- the compound Ig obtained above was used as a reagent for Clear Native electrophoresis below.
- the compound (Ig) has a 1,000-fold decrease in the absorption of 582 nm light (absorption maximum of CBB G250) compared to CBB G250.
- the 1 mg / ml aqueous solution of compound (Ig) showed almost no color visually.
- Electrophoresis system Xcell SureLock mini cell and PowerEase 500 power supply (Invitrogen)
- Cathode solution (BN-PAGE) 50 mM Tricine, 7.5 mM imidazole, and 0.02% (W / V) CBB G250 (pH 7.0)
- CN-PAGE 50 mM Tricine, 7.5 mM imidazole, and 0.02% (W / V) Compound (Ig) (pH 7.0)
- Anode solution 25 mM imidazole (pH 7.0)
- BN-PAGE a sample was prepared by dissolving a protein sample (20 ⁇ g) in a solution containing 50 mM imidazole, 50 mM NaCl, 0.5% dodecyl maltoside, and 0.5% CBB G250.
- CN-PAGE a sample was prepared in the same manner as described above except that compound (Ig) was used
- the gel used was polyacrylamide gel (Native PAGE TM 4-16% Bistris gel, 1.0 mm, 10 wells, Invitrogen).
- Native electrophoresis using CBB G250 is also called Blue Native PAGE (or BN-PAGE).
- Native electrophoresis using compound (Ig) is also referred to as ClearClNative PAGE (or CN-PAGE).
- the sample was loaded into the well, and electrophoresis was performed by applying a voltage at 150 V for 1 hour and further at 250 V for 1 hour. After electrophoresis, in the case of BN-PAGE, it was decolorized with 30% (V / V) methanol-10% (V / V) acetic acid aqueous solution. In the case of CN-PAGE, staining was performed with Imperial® Stain (Pierce).
- Protein samples are water soluble protein (Soluble protein), thyroglobulin (Thyroglobulin, molecular weight 669 kDa), ferritin (Ferritin, molecular weight 440 kDa), aldolase (Aldolase, molecular weight 158 kDa), conalbumin (molecular weight 75 kDa), ovalbumin ( Ovalbumin, molecular weight 43 kDa), carbonic anhydrase (Carbonic Anhydrase, molecular weight 29 kDa), ribonuclease A (RNase A, molecular weight 13.7 kDa) were used.
- Soluble protein Soluble protein
- Thyroglobulin Thyroglobulin, molecular weight 669 kDa
- ferritin Feritin, molecular weight 440 kDa
- aldolase Aldolase, molecular weight 158 kDa
- conalbumin molecular weight 75
- nitric oxide reductase (NOR, molecular weight 68.5 kDa), band 3 (Band3, molecular weight 55 kDa (110 kDa)), and adenosine receptor A2a (A2a, molecular weight 33 kDa) were used as membrane proteins.
- CN-PAGE showed almost the same pattern as BN-PAGE for both water-soluble proteins and membrane proteins.
- the compound (Ig) binds to the protein in the same manner as CBB G250 and can be used for electrophoresis.
- the cathode buffer is dark blue, so it was difficult to determine whether the sample was loaded properly and whether there was any leakage from the well when loading the sample into the well.
- the cathode buffer was transparent, this was not the case, and it was very easy to apply the sample to the gel.
- EGFP was used as the fluorescent protein.
- EGFP was prepared by budding yeast as a host, expressed using an EGFP expression plasmid, and purified using Ni-NTA super flow (Qiagen).
- a fusion protein of adenosine receptor A2a and EGFP (hereinafter referred to as membrane protein-EGFP fusion) was used.
- Membrane protein-EGFP fusion is expressed using plasmid DNA encoding A2a-EGFP fusion using Saccharomyces cerevisiae as a host. After cell recovery, the cells are disrupted with glass beads, and the membrane fraction is obtained by ultracentrifugation. Was recovered and prepared.
- the membrane fraction of Saccharomyces cerevisiae containing EGFP (75 ng) and membrane protein-EGFP fusion (about 50 ng) prepared above was solubilized with 1% dodecyl maltoside and electrophoresed with compound (Ig).
- the gel was photographed with excitation light of 470 nm LED and 515 nm low-pass fluorescent filter.
- the electrophoresis conditions were the same as those described in 3 above. The result is shown in FIG.
- purified EGFP was only slightly detectable by CBB staining, but could be clearly detected by fluorescence imaging.
- membrane protein-GFP fusion in CBB staining, it is not possible to identify the band hidden behind other large amounts of coexisting proteins, but the molecular weight and association state can be clearly detected by fluorescence imaging. It was.
- complicated analysis operations such as Western blotting, purification, and gel filtration are necessary. However, by using this method, analysis can be performed very easily and in a short time. It turns out that it will be possible.
- Electrophoresis was the same as the above 3 conditions. After electrophoresis, the gel was colored by incubating with 0.01% nitro blue tetrazolium, 0.05% X-gal PBS, pH 7.0 for 20 minutes.
- FIG. 4 The results are shown in FIG.
- the left side of FIG. 4 shows the gel after BN-PAGE.
- the right side of FIG. 4 shows the gel after CN-PAGE.
- the amount of ⁇ -galactosidase applied was the same in both gels: 10 ⁇ g, 5 ⁇ g, 2.5 ⁇ g, 1.25 ⁇ g, 1 ⁇ g, 0.5 ⁇ g, 0.25 ⁇ g, 0.125 ⁇ g from the right lane.
- Silver staining CN-PAGE was performed using membrane proteins and water-soluble proteins as protein samples, and silver staining was performed after electrophoresis.
- NOR Nitrogen monoxide reductase derived from Pseudomonas aeruginosa
- molecular weight 68.5 kDa was used as the membrane protein.
- NOR was prepared by culturing Pseudomonas aeruginosa, collecting the membrane fraction, collecting the membrane fraction by ultrasonic disruption, solubilizing with a surfactant, and purifying by chromatography.
- bovine-derived serum albumin (BSA, molecular weight 66 kDa, manufactured by Sigma) was used.
- the electrophoresis conditions are the same as those described in 3 above, except that the dodecyl maltoside in the sample buffer is 0.02%, the compound (Ig) is 0.05%, and the compound (Ig) in the cathode buffer is 0.002%. It was.
- Silver Staining II Kit Wako manufactured by Wako Pure Chemical Industries, Ltd.
- FIG. 5 (A) shows the results of the NOR sample.
- FIG. 5 (B) shows the result of the BSA sample.
- M on the horizontal axis indicates a marker.
- the amount of protein subjected to electrophoresis starts from the next lane of the marker and is 1000 ng, 500 ng, 100 ng, 50 ng, 10 ng, 5 ng, 1 ng, 0.5 ng, and 0.1 ng from the left.
- FIG. 5 (B) not only a band of BSA monomer (BSA) monomer) but also a band composed of several association states due to the interaction between BSA molecules was detected.
- BSA BSA monomer
Abstract
Description
項1. 中性の電気泳動緩衝液中でタンパク質と結合して分子全体がマイナスに帯電した複合体を形成することができ、かつ、実質的に無色である少なくとも1種の化合物からなる電気泳動用試薬。
項2. 前記化合物が、少なくとも1つのアリールスルホン酸部分を有する、項1に記載の電気泳動用試薬。
項3. 中性の電気泳動緩衝液中でタンパク質と結合して分子全体がマイナスに帯電した複合体を形成することができ、かつ、実質的に無色である少なくとも1種の化合物からなり、前記化合物が、下記式(I)
Ra、Rb、Rcは、同一または異なって、SO3Hもしくはその塩、ハロゲン原子、アルキル、アルケニル、アルキニル、アルコキシ、メチレンジオキシ、ヒドロキシ、トリフルオロメトキシ、トリフルオロエトキシ、トリフルオロメチル、シアノ、ニトロ、アミノ、モノアルキルアミノ、ジアルキルアミノ、アルコキシカルボニルアミノ、カルバモイル、モノ若しくはジアルキルカルバモイル、スルファモイル、モノ若しくはジアルキルスルファモイル、アルキルスルホニルアミノ、アルコキシカルボニル、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、アシルアミノまたはカルボキシルを意味し、
n1,n2、n3は、同一または異なって0~4の整数を示す。
で表される、項1又は項2に記載の電気泳動用試薬。
項4. 下記一般式(Ia)で表される少なくとも1種の化合物を含有することを特徴とする項1~3のいずれかに記載の電気泳動用試薬。
項5. 下記式(Ib)で表される少なくとも1種の化合物を含有することを特徴とする、項1~4のいずれかに記載の電気泳動用試薬。
Ra、Rcは、同一または異なって、水素原子もしくはメチルである。
項6. 下記式(Ic)で表される少なくとも1種の化合物を含有することを特徴とする、項1~5のいずれかに記載の電気泳動用試薬。
項7. 電気泳動用ゲルと項1~6のいずれかに記載の電気泳動用試薬を含む電気泳動用ゲル組成物。
項8. 電気泳動用バッファーと項1~6のいずれかに記載の電気泳動用試薬を含む電気泳動用バッファー組成物。
項9. 項1~6のいずれかに記載の電気泳動用試薬をタンパク質サンプルおよび/または電気泳動用バッファーに添加してタンパク質のClear Native電気泳動を行うことを特徴とするタンパク質の分離方法。
項10. 項1~6のいずれかに記載の電気泳動用試薬、項7に記載の電気泳動用ゲル組成物、項8に記載の電気泳動用バッファー組成物からなる群から選ばれる少なくとも1種を含むことを特徴とするタンパク質の電気泳動用キット。
項11. 下記式(Ig)、(Ih)のいずれかで表される化合物。
<スキーム1>
を有する酸性染料(スルホ基またはその塩を1~6個有する染料)を原料とし、これを必要に応じて誘導体化し、NaCNBH3などの還元剤で還元することにより、実質的に無色の以下の部分構造
を有する本発明の化合物を得ることができる。
<スキーム2>
カソードバッファー: 25mM トリスベース, 192mM グリシン, pH 8.5
アノードバッファー: 25mM トリスベース, 192mM グリシン, pH 8.5
の組成のものが挙げられる。
カソードバッファー: 7.5mM イミダゾール, 50mM トリシン, pH7.0
アノードバッファー: 25mM イミダゾール, pH 7.0
の組成のものが挙げられる。
カソードバッファー: 50mM ビス-トリス, 50mM トリシン, pH 6.8
アノードバッファー: 50mM ビス-トリス, 50mM トリシン,pH 6.8
の組成のものが挙げられる。
0 ℃で、ベンゼンメタンアミニウム, N-[4-[[4-[(4-エトキシフェニル)アミノ]フェニル][4-[エチル[(3-サルフォフェニル)メチル]アミノ]フェニル]メチレン]-2,5-シクロヘキサジエン-1-イリデン]-N-エチル-3-サルフォ-,ヒドロキサイド, インナーソルト, モノソディウムソルト
(Benzenemethanaminium,N-[4-[[4-[(4-ethoxyphenyl)amino]phenyl][4-[ethyl[(3-sulfophenyl)methyl]amino]phenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl-3-sulfo-, hydroxide, inner salt, monosodium salt)(CBB G250) 12.0 g(14.1 mmol) に、メタノール (60 mL)、シアノ水素化ホウ素ナトリウム(sodium cyanoborohydride)2.65 g(42.3 mmol) を加え、室温で 1.5 時間攪拌した。
(以下、化合物Ig) 8.95 g(収率73%)を得た。
1H NMR (270 MHz, CD3OD) : δ7.65 -7.85 (m, 4H), 7.37 (brs, 4H), 7.00 (d, J = 9.2Hz, 2H) 6.70 -6.90 (m, 6H), 6.35 -6.57 (m, 6H), 5.36 (s,1H), 4.50 (s,4H), 3.97 (q,J = 6.6 Hz,2H), 3.43 (q,J= 6.6 Hz,4H), 2.03 (s,6H), 1.34 (t,J= 6.6 Hz,3H), 1.15 (t,J = 6.6 Hz,6H).
また上記反応式を下記に示す。
上記で得られた化合物(Ig)及び市販のCBB G250(ナカライテスク株式会社)を水に溶解し、分光光度計(Hitachi UV-2810)を用いて、可視吸収スペクトルを測定した。
ゲル濾過分子量マーカー(GEヘルスケア)と3種類の膜タンパク質を対象とし、化合物(Ig)とCBB G250を用いたNative PAGEのパターンの比較を行った。
(1)電気泳動システム
Xcell SureLock mini cell及びPowerEase 500 power supply(Invitrogen社)
(2)カソード溶液
(BN-PAGE)
50mM トリシン, 7.5mM イミダゾール、及び0.02%(W/V) CBB G250(pH7.0)
(CN-PAGE)
50mM トリシン, 7.5mM イミダゾール、及び0.02%(W/V) 化合物(Ig)(pH7.0)
(3)アノード溶液
25mM イミダゾール(pH 7.0)
サンプルは、BN-PAGEの場合は、タンパク質試料(20μg)を、50mM イミダゾール, 50mM NaCl, 0.5%ドデシルマルトシド, 及び0.5% CBB G250を含む溶液に溶解して調製した。CN-PAGEの場合は、CBB G250に代えて化合物(Ig)を用いる以外は前記と同様にしてサンプルを調製した。
蛍光タンパク質と膜タンパク質の融合タンパク質を形成して、化合物(Ig)を用いたCN-PAGEを行った後、蛍光検出を行った。
大腸菌由来β-ガラクトシダーゼを用いて、BN-PAGE及びCN-PAGEを行い、ゲル中での酵素活性の検出を行った。
膜タンパク質及び水溶性タンパク質をタンパク質試料として、CN-PAGEを行い、電気泳動後、銀染色を行った。
Claims (9)
- 中性の電気泳動緩衝液中でタンパク質と結合して分子全体がマイナスに帯電した複合体を形成することができ、かつ、実質的に無色である少なくとも1種の化合物からなり、前記化合物が、下記式(I)
R’は、水素原子、SO3Hもしくはその塩、ハロゲン原子、アルキル、アルケニル、アルキニル、アルコキシ、メチレンジオキシ、ヒドロキシ、トリフルオロメトキシ、トリフルオロエトキシ、トリフルオロメチル、シアノ、ニトロ、アルコキシカルボニルアミノ、カルバモイル、モノ若しくはジアルキルカルバモイル、スルファモイル、モノ若しくはジアルキルスルファモイル、アルキルスルホニルアミノ、アルコキシカルボニル、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、カルボキシルまたはNR3R4を示す。
R”は、水素原子、SO3Hもしくはその塩、ハロゲン原子、アルキル、アルケニル、アルキニル、アルコキシ、メチレンジオキシ、ヒドロキシ、トリフルオロメトキシ、トリフルオロエトキシ、トリフルオロメチル、シアノ、ニトロ、アルコキシカルボニルアミノ、カルバモイル、モノ若しくはジアルキルカルバモイル、スルファモイル、モノ若しくはジアルキルスルファモイル、アルキルスルホニルアミノ、アルコキシカルボニル、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、カルボキシルまたはNR5R6を示す。
R1,R2,R3,R4,R5,R6は、同一または異なって水素原子、アルキル、シクロアルキル、置換されていてもよいアリールまたは置換されていてもよいアラルキルを示し、
Ra、Rb、Rcは、同一または異なって、SO3Hもしくはその塩、ハロゲン原子、アルキル、アルケニル、アルキニル、アルコキシ、メチレンジオキシ、ヒドロキシ、トリフルオロメトキシ、トリフルオロエトキシ、トリフルオロメチル、シアノ、ニトロ、アミノ、モノアルキルアミノ、ジアルキルアミノ、アルコキシカルボニルアミノ、カルバモイル、モノ若しくはジアルキルカルバモイル、スルファモイル、モノ若しくはジアルキルスルファモイル、アルキルスルホニルアミノ、アルコキシカルボニル、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、アシルアミノまたはカルボキシルを意味し、
n1,n2、n3は、同一または異なって0~4の整数を示す。
Zは、水素原子、ハロゲン原子、OH、アルコキシ、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、SH、アルキルチオ、アルキル、アミノ(NH2)、モノアルキルアミノ、ジアルキルアミノまたはアシルアミノを示す。
ただし、式(I)の化合物は、1,2,3,4,5または6個のSO3Hまたはその塩を有する。)
で表される、電気泳動用試薬。 - 下記一般式(Ia)で表される少なくとも1種の化合物を含有することを特徴とする請求項1に記載の電気泳動用試薬。
Ra、Rb、Rc、R2a、R4a、R6a、は、同一または異なってSO3Hもしくはその塩、ハロゲン原子、アルキル、アルケニル、アルキニル、アルコキシ、メチレンジオキシ、ヒドロキシ、トリフルオロメトキシ、トリフルオロエトキシ、トリフルオロメチル、シアノ、ニトロ、アミノ、モノアルキルアミノ、ジアルキルアミノ、アルコキシカルボニルアミノ、カルバモイル、モノ若しくはジアルキルカルバモイル、スルファモイル、モノ若しくはジアルキルスルファモイル、アルキルスルホニルアミノ、アルコキシカルボニル、アルキルカルボニルオキシ、アリールカルボニルオキシ、アリールオキシ、アシルアミノまたはカルボキシルを示す。
n1,n2、n3は、同一または異なって0~4の整数を示す。
m1,m2、m3は、同一または異なって0~5の整数を示す。
ただし、式(Ia)の化合物は、1,2,3,4,5または6個のSO3Hまたはその塩を有し、かつ、R1,R3,R5,
- 下記式(Ib)で表される少なくとも1種の化合物を含有することを特徴とする、請求項1または2に記載の電気泳動用試薬。
Ra、Rcは、同一または異なって、水素原子もしくはメチルである。
R2b、R2cは、一方が水素原子であり、他方がSO2NR9R10、SO3Hまたはその塩を示す。
R4b、R4cは、一方が水素原子であり、他方がアルコキシを示す。
R6b、R6cは、一方が水素原子であり、他方がSO2NR9R10、SO3Hまたはその塩を示す。
R9,R10は、水素又はアルキルを示す。
ただし、R2b、R2c、R6b、R6cの1つ又は2つは、SO3Hまたはその塩を示す。) - 電気泳動用ゲルと請求項1~4のいずれかに記載の電気泳動用試薬を含む電気泳動用ゲル組成物。
- 電気泳動用バッファーと請求項1~4のいずれかに記載の電気泳動用試薬を含む電気泳動用バッファー組成物。
- 請求項1~4のいずれかに記載の電気泳動用試薬をタンパク質サンプルおよび/または電気泳動用バッファーに添加してタンパク質のClear Native電気泳動を行うことを特徴とするタンパク質の分離方法。
- 請求項1~4のいずれかに記載の電気泳動用試薬、請求項5に記載の電気泳動用ゲル組成物、請求項6に記載の電気泳動用バッファー組成物からなる群から選ばれる少なくとも1種を含むことを特徴とするタンパク質の電気泳動用キット。
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