CN101473228B - Use dyestuff and the protein detection reagents of dextrin and method - Google Patents
Use dyestuff and the protein detection reagents of dextrin and method Download PDFInfo
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- CN101473228B CN101473228B CN200780021434.8A CN200780021434A CN101473228B CN 101473228 B CN101473228 B CN 101473228B CN 200780021434 A CN200780021434 A CN 200780021434A CN 101473228 B CN101473228 B CN 101473228B
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- dextrin
- detergent
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 96
- 229920001353 Dextrin Polymers 0.000 title claims abstract description 82
- 239000004375 Dextrin Substances 0.000 title claims abstract description 82
- 235000019425 dextrin Nutrition 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 54
- 239000000975 dye Substances 0.000 title claims description 104
- 238000002331 protein detection Methods 0.000 title claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 116
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 116
- 239000003599 detergent Substances 0.000 claims abstract description 51
- 239000002131 composite material Substances 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 35
- 239000002253 acid Substances 0.000 claims description 34
- 229920000858 Cyclodextrin Polymers 0.000 claims description 25
- 230000008859 change Effects 0.000 claims description 21
- 239000000499 gel Substances 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 18
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 230000015572 biosynthetic process Effects 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 10
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 9
- 238000013139 quantization Methods 0.000 claims description 9
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 8
- 239000004094 surface-active agent Substances 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- -1 (methylene) tri methylene phosphonic acid Chemical compound 0.000 claims description 6
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000000295 emission spectrum Methods 0.000 claims description 5
- 150000003009 phosphonic acids Chemical class 0.000 claims description 5
- 229920002401 polyacrylamide Polymers 0.000 claims description 5
- 239000001045 blue dye Substances 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 3
- 239000011543 agarose gel Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 229960005215 dichloroacetic acid Drugs 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 230000005684 electric field Effects 0.000 claims description 3
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 3
- 239000011976 maleic acid Substances 0.000 claims description 3
- 235000006408 oxalic acid Nutrition 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- QYHFIVBSNOWOCQ-UHFFFAOYSA-N selenic acid Chemical compound O[Se](O)(=O)=O QYHFIVBSNOWOCQ-UHFFFAOYSA-N 0.000 claims description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 3
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 2
- 238000002835 absorbance Methods 0.000 claims 4
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims 1
- 239000002552 dosage form Substances 0.000 claims 1
- 239000000835 fiber Substances 0.000 claims 1
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- 229910052740 iodine Inorganic materials 0.000 claims 1
- 239000011630 iodine Substances 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 239000012142 reagent concentrate Substances 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 12
- 239000000243 solution Substances 0.000 description 35
- 239000000523 sample Substances 0.000 description 26
- 239000000203 mixture Substances 0.000 description 24
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 15
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 15
- 230000009182 swimming Effects 0.000 description 12
- 102000008192 Lactoglobulins Human genes 0.000 description 10
- 108010060630 Lactoglobulins Proteins 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 10
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 10
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 9
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 239000004141 Sodium laurylsulphate Substances 0.000 description 8
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 8
- 238000002731 protein assay Methods 0.000 description 7
- 239000011546 protein dye Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 description 5
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 description 5
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- 229940043377 alpha-cyclodextrin Drugs 0.000 description 5
- 239000012152 bradford reagent Substances 0.000 description 5
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 5
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
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- 206010053567 Coagulopathies Diseases 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 241000047703 Nonion Species 0.000 description 2
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
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- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 2
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- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical class O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 2
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical class O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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Abstract
The present invention is provided to detect protein and quantitatively determine the reagent of protein concentration, method and test kit.Described reagent includes protein composite dye, such as Coomassie dye and one or more dextrin, thus eliminates the interference caused by detergent.
Description
Technical field
The present invention relates to for detecting protein and quantitatively determining the reagent of protein concentration, method and test kit.
Background technology
There is many for detecting protein and the method determining concentration of protein in solution.These include well known in the art
Dyestuff-associated methods, and include nonspecific reaction, in the reaction, protein-complexing dye and protein bound.Dye
The formation of material-protein complex causes the change of dyestuff optical property, thus also exist with sample present in protein
Measure proportional color change.The protein-complexing dye quantified for external protein includes bromocresol green (Gindler, U.S.
State's patent No. 3,884,637), HABA and methyl orange, but be incorporated into albumin due to them the most exclusively and be frequently not
The sensitiveest, so having the application of limitation.
Other determine that the method for protein concentration includes biuret (Biuret) method (Mokrasch and McGilvery, life
Thing and The Chemicals (J.Biol.Chem.) (1956) .221, page 909), wherein contain the peptide structure of at least two peptide bond
With the Cu in alkaline solution2+Reaction, thus form purple chelate.
Lowry etc. (laboratory clinical medical journal (J.Lab.Clin.Med.) (1951) .39,663) employ and utilize alkali
The protein pretreatment of property copper solution, is similar to biuret method, adds forint-Theo Ka Te (Folin-subsequently
Ciocalteu) reagent (it contains the lithium salts of phosphotungstic acid and phosphomolybdic acid).The color produced is by Cu-protein complex with by this
Phosphotungstic acid and phosphomolybdic acid are reduced to the result of tungsten and molybdenum blue by tryptophan and the tyrosine of protein.
The critical defect of biuret and Lowry method is that they can not ever-present reduction in tolerance protein quality sample
Agent.
Have been described with utilizing Coomassie brilliant G-250 to form dyestuff/protein complex as protein-complexing dye
(Bradford U.S. Patent number 4,023,933).Coomassie Brilliant Blue dye and huge variety of protein bound.And, use
The G-250 being in suitable acid medium causes such protein assay reagents, described protein assay reagents to have
Biuret and about 100 times of Conventional dye combination technology, be that (Bradford is beautiful for the sensitivity of about 3-5 times of Lowry method
State's patent No. 4,023,933).Coomassie brilliant G-250, i.e. ＂ is used in the U.S. 4, the program disclosed in 023,933
Bradford measures ＂, has many and surmounts the advantage using other dye methods, including high sensitivity, this allow that and work as sample
In when there is reducing agent, use sample size and effectiveness.
Coomassie brilliant G-250 is with two kinds of different colored forms, and i.e. red and blueness exists.The blue form of this dyestuff
Occur in neutrality and alkaline solution, and red form occurs in the most acid solution (pH0-1).In an acidic solution,
Coomassie brilliant G-250 is in the poised state of red and blue form；Such solution is the most brown.Think and work as egg
White matter is when dyestuff is combined, and dyestuff is introduced in different microenvironments, and is then subjected to protection in order to avoid meeting with and carrying for this dyestuff
Acid medium for red color.The intensity of acid medium is the heaviest for the sensitivity utilizing the protein determination of Coomassie dye
Want, because increasing of acid medium intensity causes significantly reducing of this mensuration sensitivity.Protein-dye complex tends to gather
Collection, this affects the stability of color product.Solubilizing agent, the existence of such as ethanol tends to keep albumen within rational period
Matter-dye composition is not assembled；But, too many ethanol causes the notable skew to this dyestuff blueness form, will change by environment
Become less polarity.Assuming that the mechanism of this mensuration is the combination of dyestuff carbanion and the less polar environment of protein.This can
A large amount of detergent and the acetone negative interaction to this mensuration can be also explains, because these compounds are typically natural non-polar,
And should tend to change the environment of this dyestuff.
The major defect that Bradford measures is substantial lack colour stability in prolonging period, and this is mainly due to
The precipitation of protein-dye complex；The most do not demonstrate the reactivity identical to different proteins；Disobey Beer rule；
Most significantly, detergent detrimental effect (Bradford, M., analytical biochemistry to this mensuration present in sample
(Anal.Biochem.), 72 248-254,1976 and U.S. Patent number 4,023,933).
The formation of dyestuff/protein complex is additionally operable to the protein in gel, and those used in such as electrophoresis are entered
Row dyeing.Such as, dyestuff Coomassie brilliant G-250 (Reisner, the A.H. etc. being in perchloric acid solution are so employed
(1975) analytical biochemistry (Anal.Biochem.) 64,509-516).
At present, many comls, preparation based on coomassie may be used for after electrophoretic separation in gel
Protein dyes.For many electrophoresis application, detergent such as SDS is used to promote the separation of protein.Because detergent
Negatively affect the color change caused due to Coomassie dye and protein bound, so must be gone by some cleaning procedures
Except detergent, this causes tediously long and complicated dyeing procedure.
Therefore, protein detection based on dyestuff and quantization, measure reagent or Coomassie dye in particular with Lowry
Major defect is the interference from detergent, surfactant and other amphipathic molecules.
Accordingly, there exist the reagent and the needs of method to such detection and quantitatively determining protein, described reagent and
Method has the toleration of raising to there is detergent in sample, and has the protein-dye colour stability of raising.
Summary of the invention
The present invention is provided to detect the reagent of protein, it includes or is made up of the following:
(a) protein-complexing dye, and
(b) one or more dextrin.
Described protein-complexing dye is such dyestuff, and it is typically due to form protein-dye complex and warp
Going through the change of optical property, this can be as used coomassieTMLight blue dyestuff, bromocresol green, HABA, methyl orange, biuret try
Agent, the change having in the absorption spectrum that the biuret reagent (Lowry reagent) of forint-Theo card spy's reagent occurs；Or as right
Form the dyestuff of fluorescin/dye composition, such as coomassie orangeTM, fluorescein, Alexofluor, phycoerythrin, De Kesa
This is redTM(Texas RedTM) change in the emission spectrum that occurs.
Preferably, described protein-complexing dye does not include protein, it is preferable that described protein-complexing dye does not wraps
Include antibody or peptide.
Described protein-complexing dye is preferably Coomassie dye, such as Coomassie Brilliant Blue dye, and such as coomassie is bright
Blue dyestuff G-250 or Coomassie Brilliant Blue dye R-250.For some protein composite dyes, and particularly Coomassie dye, need
Will low pH, thus obtain the necessary change of optical property when the formation of protein/dye composition.
Therefore, the present invention also provides for the reagent for detecting protein, comprising:
(a) protein-complexing dye,
(b) one or more dextrin, and,
C () has the acid of 4 or lower pKa.
In described reagent, it is preferable that described dyestuff is with about 0.001%-about 0.1% (w/v), preferably about 0.005%-
Concentration in the range of 0.05% (w/v) exists.In use, described reagent, typically, reagent and diluent, example can be diluted
Should be in the range of about 1:1-about 1:60 as wrapped the ratio of proteinaceous solution.In order to detect containing 25 μ g/ml or more
Protein in the solution of few protein, it is possible to use the reagent of 1:1 and the volume ratio wrapping proteinaceous solution.For tool
There are the solution of more increased protein concentration, such as, 0.1mg/ml-2mg/ml, reagent and the volume ratio 1 wrapping proteinaceous solution:
60 should be suitable.
In the range of useful acid has and is in 0-4, the pKa of preferably 3 or lower, so that described reagent has-1 to 1
pH；It is highly preferred that described acid should have the pKa being in the range of about 1-about 3, so that described reagent has the pH of 0-1.?
Bradford patent (US 4,023,933) and Gindler patent (US 4,239,495) determine much useful acid, closes
Suitable acid includes phosphoric acid, phosphorous acid (phosphonic acids), periodic acid, selenic acid, maleic acid, oxalic acid and dichloroacetic acid.Phosphoric acid and phosphonic acids are excellent
Choosing.Preferably phosphonic acids is time amino three (methylene) tri methylene phosphonic acid (Nitrilotris (methylene) triphosphonic
Acid, NTP), it is a kind of commercially available polyprotic acid.
According in the reagent of the present invention, in the presence of acid, generally with about 4%-about 20%, it is preferable that about 4%-is about
12%, it is preferable that the concentration of about 7.5%-about 9.5% (w/v) exists.In use can dilute described reagent, so acid
Ultimate density is in the range of about 2%-about 20%.
Described acid can be polynary and monacid mixture, in described mixture, it is preferable that polynary and monacid
Ratio is in the range of about 2:1-about 3:1.In described reagent, polynary/monoacid mixture generally with about 1-about 15% (v/v),
Preferably, the concentration in the range of about 2%-about 5% (v/v) exists.In order to use, described reagent can be diluted, thus at offer
The ultimate density of the polynary/monoacid mixture in the range of about 0.5-about 15%.
Include that one or more dextrin, described dextrin are preferably chosen from linear dextrin (D), ring is stuck with paste according to the reagent of the present invention
Essence (CD), cycloamylose (CA) and their derivant.Preferably reagent includes one or more cyclodextrin.Suitably line
Property dextrin includes 6 or more glucose unit, it is preferable that 10 or more glucose unit, such as 15 glucoses
Unit；Cyclodextrin is generally of 6 (α-CD), 7 (β-CD) or 8 (γ-CD) glucose units；Cycloamylose leads to
Often include 8 or more glucose unit.Suitably derivant includes heptakis2,6-bis--o-butyl beta-schardinger dextrin-, carboxylic
Methyl beta-cyclodextrin and carboxymethyl alpha-cyclodextrin.
The mixture of the mixture of dextrin, such as cyclodextrin can be used, the most two or more selected from α-CD, β-CD
Mixture with the cyclodextrin of γ-CD；The mixing of the two or more cyclodextrins selected from α-CD, β-CD, γ-CD and CA
Thing；The mixing of one or more linearly and in the mixture of cyclodextrin, such as linear dextrin and α-CD, β-CD and γ-CD
Thing；Or linear dextrin and the mixture of one or more in α-CD, β-CD, γ-CD and CA.Unless context it is further noted that
Term " dextrin " is for herein time, including dextrin and dextrin derivative.
Some derivants of cyclodextrin, dextrin and some cycloamylose can play the effect of surfactant, and
And may more not be suitable in the reagent of the present invention, method and test kit.Some are in the dextrin under finite concentration, due to this
The surface-activation character of dextrin, will disturb some dyestuff.Those skilled in the art can be readily determined described dextrin and it
Interference concentration respective to different dyes.
For given protein, the selection of the dextrin used or cyclodextrin mixtures can be carried out optimization, and
When using one or more dextrin, ratio can be regulated, thus obtain detection and/or quantify given protein or albumen
The maximally effective condition of quality sample.
In the reagent of the present invention, in the range of dextrin is generally with 0.01-200mg/ml, it is preferable that 0.5-50mg/ml scope
Interior concentration exists.When using cyclodextrin mixtures, these concentration relate to total dextrin concentration.The dilute of described reagent can be regulated
Release, so that final dextrin concentration is optimum for given protein and protein concentration.When protein example exists decontamination
During agent, can be for the selection of given detergent and the concrete concentration optimization dextrin of this detergent and concentration.This area skill
Art personnel, such as, pass through in the presence of the dextrin or cyclodextrin mixtures of variable concentrations, in due course, and measurement protein-
The absorption of dye composition or emission spectrum, it is possible to easily determine the most final dextrin concentration.For Coomassie brilliant blue G-
250, it is possible at absworption peak, i.e. measure at 595nm and absorb.
Solubilizing agent, such as alcohol can also be included according to the reagent of the present invention, thus maintain the molten of dye-protein matter complex
Xie Xing.Described solubilizing agent can be any minimizing or the reagent postponing dye-protein matter complex precipitate.
Can include one or more alcohol in described reagent, suitable alcohol includes ethanol, methanol and propanol.Other are suitable
Alcohol is those with good water-soluble, and it act as detergent hardly or not.When there is alcohol in described reagent, it is dense
Degree is typically 0.1%-about 10% (v/v), it is preferable that about 0.1%-about 5% (v/v), it is highly preferred that about 1%-about 5% (v/
v)。
The reagent of the present invention can include detergent.
Such as can provide institute's reagent as one or more aqueous components with many part system, described aqueous components is tied
Close the reagent forming the present invention.If provided with two parts, then a part can include described dyestuff, optionally, acid and/or
Optionally, alcohol, and another part can include described dextrin.Each single component has long-term stability and (works as freezing
During preservation, about 1 year), and when being mixed to form described reagent mutually, this reagent from be frozen be saved in 4 DEG C time, keep stable
More than 6 months.By making one or more dextrin and commercially available protein staining agent, such as Bradford measure reagent or its
He combines by coomassie protein stain, can generate the reagent according to the present invention.By mixing one or more according to this
Dextrin described in invention, it is possible to transform commercially available protein staining agent, method and test kit；The most commercially available
Bradford measures reagent, method and test kit.The example of described commercially available test kit includes the following:
Pierce:
23236 coomassies add (Coomassie Plus)-more preferable Bradford and measure test kit (TheBetter
Bradford Assay Kit) (including reference material)
23238 coomassies add-more preferable Bradford mensuration reagent
23200 coomassies (Bradford) Protein Assay Kit
23296 coomassies (Bradford) dried protein assay plate 2x96 hole
23596 coomassies (Bradford) dried protein assay plate 5x96 hole
BioRad:
500-0201EDU quickly initiates Bradford Protein Assay Kit (Quick StartBradford
Protein Assay Kit)1
500-0202EDU quickly initiates Bradford Protein Assay Kit 2
500-0203EDU quickly initiates Bradford Protein Assay Kit 3
500-0204EDU quickly initiates Bradford Protein Assay Kit 4
500-0006EDU Bio-Rad protein assay dye reagent concentrates (Bio-Rad ProteinAssay Dye
Reagent Concentrate)
Sigma-Aldrich (Sigma Aldrich):
B6916 Bradford reagent (Sigma (Sigma))
27813 coomassiesProtein assay reagents BioChemika (Fluka)
Routinely, the detection of some protein-complexing dye and quantization protein is utilized to be subjected to from detergent very
Significantly interference；It is protein composite dye Coomassie brilliant blue G-250, the red G-250 of coomassie by particularly disadvantageous impact, is examined horse
This orange, biuret reagent and there is the biuret reagent of forint-Theo card spy's reagent.Instant invention overcomes these difficult problems.Not
Under conditions of wishing to be bound by theory, it is believed that described detergent forms complex with described dextrin, and described detergent is to institute
State the affinity of dextrin higher than the described detergent affinity to described dyestuff.By using proper amount of dextrin or dextrin to mix
Thing, described detergent can be captured by dextrin-detergent complex, thus, limits the suppression protein-dye reaction of this detergent
Degree.
Compared with the reagent being presently available for protein detection, when there is detergent in the proteinaceous sample of bag, energy
Enough successfully use the reagent of the present invention.This has the most great meaning, because the reagent of the present invention is allowed in rich in detergent
Or the environment of surfactant detects protein, described detergent or surfactant can be such as that solubilising memebrane protein is musted
Need, or utilize the solution rich in detergent, the most commercially available extraction solution, such as, and CelLyticTMDirectly
Extract necessary to protein from microorganism.
The present invention also provides for detecting method of protein, and it includes making the proteinaceous sample of bag and comprises the following
Solution contacts:
(a) protein-complexing dye, and,
(b) one or more dextrin,
Formation with detection dyestuff/protein complex.
For needing the dyestuff of strong acidic condition, the invention provides detection method of protein, it includes making to comprise egg
The sample of white matter contacts with the solution comprising the following:
(a) protein-complexing dye,
(b) one or more dextrin, and,
C () has the acid of 4 or lower pKa；
Formation with detection dyestuff/protein complex.
The formation of detection dyestuff/protein complex can include the amount of dyestuff/protein complex quantifying to be formed,
Thereby determine that the protein concentration in described sample.
In another embodiment, the present invention provides quantization method of protein, and it includes making the proteinaceous sample of bag
Product contact with the solution comprising the following:
(a) protein-complexing dye,
(b) one or more dextrin,
With the formation quantifying dyestuff/protein complex.
For needing the dyestuff of strong acidic environment, the present invention provides quantization method of protein, and it includes making to comprise albumen
The sample of matter contacts with the solution comprising the following:
(a) protein-complexing dye,
(b) one or more dextrin, and,
C () has the acid of 4 or lower pKa；
With the formation quantifying dyestuff/protein complex.
Wrapping proteinaceous sample can be solution, or can be at support, such as gel, colloidal sol, chromatography plate, filter
There is provided on paper, nitrocellulose membrane or resin and wrap proteinaceous sample.
Therefore, in another embodiment, the present invention also provides for detecting method of protein, comprising:
A () provides the support including protein,
B () makes described protein and contacts with the solution including the following:
(i) protein-complexing dye, and,
(ii) one or more dextrin
Formation with detection dyestuff/protein complex.
For needing the protein complex dyestuff of acid condition, the invention provides detection method of protein, its bag
Include:
A () provides the support including protein,
B () makes described protein and contacts with the solution including the following:
(i) protein-complexing dye,
(ii) one or more dextrin, and,
(iii) there is the acid of 4 or lower pKa；
Formation with detection dyestuff/protein complex.
The foregoing describe the suitable protein composite dye used in the inventive method, dextrin and if it is required, use
In the acid mixed in described solution.Described protein-complexing dye, one or more pastes can be provided according to the reagent of the present invention
Essence and if it does, have the acid of 4 or lower pKa, the reagent of the present invention can be diluted formed solution.Described solution can
To include solubilizing agent, such as alcohol as described herein.
Described support can be gel, colloidal sol, chromatography plate, filter paper, nitrocellulose membrane or resin.Described support can
To include detergent.The method utilizing the present invention, contact can be carried out under conditions of there is detergent.These methods are special
It is not suitable for detecting the protein in polyacrylamide gel, agarose gel or polymer composite gel, such as in profit
With electric field, during protein example the most separated by electrophoresis.
Specifically described herein for detecting and quantitatively determine the protein in gel, such as utilizing electric field, such as, passing through
Reagent and the method for those protein produced after electrophoretic separation simplify conventional program, are so no longer necessary to remove detergent all
Such as SDS and the cleaning procedure of excess dyeing (background stainings).
Typically, the method is at room temperature performed.
In the method for the invention, the formation of detection dyestuff protein complex can include detecting described dyestuff/albumen
Change in the absorption of matter complex or emission spectrum.In some situation, color change can be detected；Such as examine horse when use
This light blue dyestuff, such as during G-250.Color change can utilize conventional equipment, such as, such as, can measure at 570nm-
The tintometer absorbed at wavelength in the range of 620nm.
For experienced by the dyestuff of change in absorption spectrum owing to forming protein/dye composition, detection can be led to
Cross, such as, utilize metric measurement to absorb and realize.Conventional equipment can be used to carry out spectrophotometric analysis, such as make
With the UV/VIS spectrophotometer with 400-700nm wave-length coverage.
For experienced by the dyestuff of change in emission spectrum owing to forming protein/dye composition, detection can be led to
Cross, such as, utilize the spectrofluorimeter (luminescence spectrometer) suitably with 190nm-800nm wave-length coverage, measure and launch and reality
Existing.
Detect described protein/dye composition and can include quantifying the amount of the protein/dye composition of existence, thus
Determine amount or the concentration of protein.Quantization can be by including that measuring described dyestuff/protein complex absorbs or launch light
In spectrum, the method for change realizes.Quantization can include, such as, measure color change.Spectrophotometric can be passed through as discussed, absorb
Method is measured, and can measure the change absorbed over time.Generally measure in the wave-length coverage of about 400-about 700nm and absorb.
For Coomassie brilliant G-250, measure at the wavelength of about 595nm and absorb, when this dyestuff is combined with protein
Time, it absorbs maximum.When utilizing Coomassie brilliant G-250, it is possible to by monitoring due to formation dyestuff/protein complex
The increase absorbed at the 595nm caused, and detect protein.
In order to determine protein concentration, absorption measurement can arrived or transmitting and standard figures, standard figures group or mark
Directrix curve compares.As in the embodiment shown, this result is that high reproducibility is with accurate.
The high sensitivity shown due to the reagent and method that utilize the present invention, it is possible to select low such as from about 0.1 μ g/1ml sample
The protein concentration of product.And, the less than about 2 minutes/sample of the time needed for the sensitiveest described mensuration, in contrast,
The mensuration of tradition Lowry or biuret type typically requires 30-40 minute.Therefore, the method for the present invention is highly suitable in a large number
The automatization of sample and analysis.
The present invention also provides for the test kit for detecting and/or quantify protein, and described test kit includes one or more
Dextrin.One or more dextrin and protein-complexing dye can be included for detecting and/or quantifying the test kit of protein.
It addition, test kit can include one or more acid and/or alcohol as described herein.According to the present invention for detection and/or
The test kit quantifying protein can include the reagent of the present invention, and described reagent can provide as many part system, wherein group
Split-phase is mixed to form the reagent of the present invention.
The present invention also provides for one or more dextrin, under conditions of there is detergent, enhancing protein-binding dye/
The application of the formation of protein complex.
Additionally, the present invention provides one or more dextrin, under conditions of there is detergent, reduce detergent at albumen
Matter-combination dye/protein complex formed in the application of interference.
The present invention also provides for one or more dextrin, under conditions of there is detergent, change dyestuff, such as protein-
The application of the optical property of composite dye.
Accompanying drawing explanation
Fig. 1: without the standard curve of the protein example of detergent.Sample without detergent is provided reasonably by two kinds of methods
Linear response.The slope of curve of two kinds of methods is similar with correlation coefficient, and this illustrates that the dextrin that this reagent includes does not disturbs albumen
The combination of matter-dyestuff.
Fig. 2: include the standard curve of the protein example of detergent (0.25% CTAB).Only include the reagent of dextrin
The tool slope of being and not being detergent samples or the similar slope of correlation coefficient and the linear response of correlation coefficient are provided.
Fig. 3: the protein in detection polyacrylamide gel
Each gel runs down row sample:
Swimming lane 1 molecular weight standard, Mark 12TM
Swimming lane 2 beta lactoglobulin 0.08mg/ml
Swimming lane 3 beta lactoglobulin 0.16mg/ml
Swimming lane 4 beta lactoglobulin 0.31mg/ml
Swimming lane 5 beta lactoglobulin 0.63mg/ml
Swimming lane 6 beta lactoglobulin 1.25mg/ml
Swimming lane 7 beta lactoglobulin 2.5mg/ml
Swimming lane 8 is blank
Swimming lane 9 beta lactoglobulin 5mg/ml
Swimming lane 10 is blank
Swimming lane 11 beta lactoglobulin 10mg/ml
Swimming lane 12 molecular weight standard
As described in example 3 above, with solution B stained gel B, with solution A stained gel A.Described staining solution is trained
After supporting 1 hour 45 minutes, to described gel images.
Embodiment
Embodiment 1:
Preparation Bradford reagent
47g ethanol, 85g phosphoric acid and 850g water is added in 100mg Coomassie brilliant blue (G-250).Mix this solution 20 points
Clock, so that it is guaranteed that all of component dissolve, formed include 0.01% (w/v) Coomassie brilliant G-250,4.7% ethanol (w/v) and
The reagent of 8.5% (w/v) phosphoric acid.As indicated in this specific embodiment, in this solution, add different dextrin.
Bradford measures (standard method)
The sample solution that 5 microlitres contain 0.1mg/ml-1.5mg/ml protein and/or 0.00%-0.5% detergent is inhaled
Move on in the hole of 96-hole microtitration flat board.It is added thereto to 300 microlitre Bradford reagent.Measure at 595nm and absorb.
Reduce detergent interference
This experiment employs 5 kinds of different detergents, i.e. sodium lauryl sulphate (SDS) (anion), cetyl
Trimethylammonium bromide (CTAB) (cation), tweenTM-20 (non-ions), TRITONTM-X100 (non-ion) and Brij-35
(non-ion).This experiment employs 4 kinds of different dextrin, i.e. dextrin-15 (D15), alpha-cyclodextrin (α-CD), beta-schardinger dextrin-
(β-CD) and gamma-cyclodextrin (γ-CD), also use these dextrin waits mass mixing thing (MIX).It addition, also examine not
Same cycloamylose concentration.
A) Bradford measures, containing 100mg/ml dextrin altogether or the saturated concentration of different dextrin.
Using water sample as blank, the absorption table measured at 595nm.
| Without dextrin | D15 | α-CD | β-CD | γ-CD | MIX | |
| SDS (0.5%w/v) | 0.326 | 0.007 | 0.013 | 0.089 | 0.038 | 0.047 |
| CTAB (0.25%w/v) | 0.785 | 0.024 | 0.003 | 0.067 | 0.015 | 0.020 |
| Tween 20 (0.25%w/v) | 1.027 | 0.453 | 0.134 | 0.077 | 0.344 | 0.041 |
| TRITON-X100 (0.10%w/v) | 0.677 | 0.101 | 0.034 | 0.017 | 0.022 | 0.003 |
| Brij-35 (0.50%w/v) | 0.231 | 0.035 | 0.015 | 0.019 | 0.143 | 0.018 |
B) Bradford measures, the dextrin of the 10mg/ml altogether containing different dextrin.
Using water sample as blank, the absorption table measured at 595nm.
| Without dextrin | D15 | α-CD | β-CD | γ-CD | MIX | |
| SDS (0.5%w/v) | 0.326 | 0.051 | 0.003 | 0.025 | 0.005 | 0.000 |
| CTAB (0.25%w/v) | 0.785 | 0.184 | 0.026 | 0.029 | 0.164 | 0.008 |
| Tween 20 (0.25%w/v) | 1.027 | 0.855 | 0.167 | 0.171 | 0.625 | 0.376 |
| TRITON-X100 (0.10%w/v) | 0.677 | 0.076 | 0.003 | 0.006 | 0.019 | 0.095 |
| Brij-35 (0.50%w/v) | 0.231 | 0.046 | -0.02 | 0.001 | 0.007 | 0.026 |
C) Bradford measures, the dextrin of the 1mg/ml altogether containing different dextrin.
Using water sample as blank, the absorption table measured at 595nm.
| Without dextrin | D15 | α-CD | β-CD | γ-CD | MIX | |
| SDS (0.5%w/v) | 0.326 | 0.195 | 0.010 | 0.056 | 0.079 | 0.020 |
| CTAB (0.25%w/v) | 0.785 | 0.674 | 0.163 | 0.176 | 0.603 | 0.407 |
| Tween 20 (0.25%w/v) | 1.027 | 0.999 | 0.842 | 0.803 | 0.952 | 0.947 |
| TRITON-X100 (0.10%w/v) | 0.677 | 0.248 | 0.288 | 0.040 | 0.032 | 0.003 |
| Brij-35 (0.50%w/v) | 0.231 | 0.098 | 0.003 | 0.033 | 0.040 | 0.002 |
D) Bradford measures, the cycloamylose (CA) containing variable concentrations.
Using water sample as blank, the absorption table measured at 595nm.
| Without dextrin | CA 10mg/ml | CA 1mg/ml | |
| SDS (0.1%w/v) | -0.063 | -0.072 | -0.023 |
| CTAB (0.1%w/v) | 0.412 | -0.081 | 0.003 |
| Tween 20 (0.1%w/v) | 0.462 | 0.074 | 0.352 |
| TRITON-X100 (0.1%w/v) | 0.677 | -0.024 | 0.410 |
| Brij-35 (0.1%w/v) | -0.024 | -0.025 | -0.011 |
Absorption at 595nm provides the instruction of the amount to coomassie G-250 dyestuff blueness form.In given concrete decontamination
Under agent concentration, high-selenium corn value represents high background, due to the existence of this detergent, so that the blueness detected does not represents the egg of existence
The amount of white matter-dye composition, and the most do not represent the concentration of protein.It will be seen that for the detergent of every kind of inspection,
In solution, the existence of one or more dextrin causes relatively low absorption value, and this explanation exists relatively low ambient interferences, and dextrin
Existence increase susceptiveness and the accuracy of protein detection.For given detergent, it is possible to by different dense in existence
Under conditions of dextrin of degree and combinations thereof, measure the absorption at dyestuff/protein complex absworption peak and determine dextrin and paste
The optimum selection of essence concentration.
Embodiment 2: there is the linear of standard curve under detergent conditions
In the concentration range of 0.1mg/ml-1.5mg/ml, create the dilution series of two kinds of different proteins, described two
Planting different proteins is bovine serum albumin (BSA) (Pierce, 23209) and B-IgG (IgG) (Sigma
(Sigma), I5506).CTAB is as detergent in selection, and adds it to reach concentration 0.25% (w/ in protein example
v).With containing 0.25mg/ml dextrin-15 (D15), 0.25mg/ml alpha-cyclodextrin (α-CD), 0.25mg/ml beta-schardinger dextrin-(β-
CD) and the Bradford reagent of 0.25mg/ml gamma-cyclodextrin (γ-CD) carries out Bradford mensuration, by these with utilize not
The Bradford that the Bradford reagent comprising dextrin is carried out measures and compares.
Embodiment 3: the protein in detection polyacrylamide gel
Prepare gel-colored solution
50g ethanol, 80g phosphoric acid and 850g water is added in 80mg Coomassie brilliant blue (G-250).Mix this solution 20 points
Clock, so that it is guaranteed that all of component dissolve-this is solution A.Subsequently, by by 250mg dextrin-15,250mg alpha-cyclodextrin (α-
CD), 250mg beta-schardinger dextrin-(β-CD) and 250mg gamma-cyclodextrin (γ-CD) join in 100ml solution A, make solution B.
Prepare sample and gel
In the concentration range of 0.08mg/ml-10mg/ml, prepare beta lactoglobulin (BLG) (Sigma (Sigma), L-
0130) dilution series.30 microlitre samples are diluted with 10 microlitres Nupage LDS sample buffer (Invitrogen, NP0007)
Product.Manufacture described gel duplicate, every kind of sample of 15 microlitre is loaded to every clotting glue (Nupage 10% Bis-Tris,
Invitrogen, NP0302) on.By 10 microlitre Mark12TMMolecular weight standard (Invitrogen, LC5677) is also loaded to this and coagulates
On glue.Under constant voltage (200V), in standard MES buffer, run gel 35 minutes.Subsequently, a clotting glue is immersed in
(staining solution being made up of 25ml solution A as above, its mixture containing dextrin is as follows: 2.5mg/ml for 25ml solution B
Dextrin-15 (D 15), 2.5mg/ml alpha-cyclodextrin (α-CD), 2.5mg/ml beta-schardinger dextrin-(β-CD) and 2.5mg/ml γ-ring
Dextrin (γ-CD)) in.Another clotting glue is immersed in the staining solution being made up of 25ml solution A.Staining solution is cultivated
After 1 hour 45 minutes, to gel images (Fig. 3).
Claims (45)
1. detection and/or a quantization method of protein, described method includes making protein and includes the solution of the following
Contact:
(a) protein-complexing dye, and
(b) one or more dextrin,
And detect and/or the formation of quantization dyestuff/protein complex,
The most to be detected or quantify protein with detergent or surfactant fluid, and one or more of which stick with paste
The concentration of essence is 1-100mg/ml.
2. according to the process of claim 1 wherein that described protein exists in the presence of detergent.
3., according to the method described in claim 1 or claim 2, wherein said protein is in the solution or described protein quilt
There is provided on support.
The most in accordance with the method for claim 3, wherein said support is gel, colloidal sol, chromatography plate, filter paper, nitrification fibre
Dimension film or resin.
Method the most according to claim 4, wherein said gel is polyacrylamide gel or agarose gel.
The most in accordance with the method for claim 5, wherein said polyacrylamide gel or agarose gel and described protein
Electric field has been utilized to be separated.
The most in accordance with the method for claim 3, wherein said support comprises detergent, and/or wherein exists at detergent
Under conditions of contact.
The most in accordance with the method for claim 1, wherein detect and/or quantify to include detecting described dyestuff/protein complex
Absorb or the change of emission spectrum.
The most in accordance with the method for claim 1, wherein detect and/or quantify to include detecting color change, described detection color
Change by using the absorbance at metric measurement wavelength in the range of 400 to 700nm to carry out.
The most in accordance with the method for claim 9, wherein said protein-complexing dye is Coomassie brilliant G-250, and
Absorbance is measured at 595nm wavelength.
11. in accordance with the method for claim 1, wherein quantified by measurement absorbance over time.
12. according to the method any one of claim 8 to 11, the absorbance wherein measurement arrived or emission measure and criterion numeral
Value, standard figures group or standard curve compare.
13. according to the process of claim 1 wherein that described method also includes: before described contact procedure, it is provided that include albumen
The support of matter.
14. one or more dextrin are for reducing answering of a kind of detergent interference in protein detection and/or quantization method
With, the concentration of one or more dextrin wherein said is 1-100mg/ml.
15. according to the application described in claim 14, wherein said dextrin with comprise protein-complexing dye and one or more
The dosage form of dextrin provides.
16. is Reagent Concentrate according to the application described in claim 15, wherein said reagent.
17. according to the application described in claim 15, and wherein said reagent also comprises the acid with 4 or lower pKa.
18. also comprise solubilizing agent according to the application described in claim 15, wherein said reagent.
19. do not comprise protein according to the application described in claim 15, wherein said protein-complexing dye.
20. according to the application described in claim 15, and wherein said dyestuff is Coomassie Brilliant Blue dye G-250.
21. exist with the concentration in the range of 0.001%-0.1% (w/v) according to the application described in claim 15, described dyestuff.
22. are diluted according to the application described in claim 15, described reagent, and wherein reagent and the ratio of diluent are 1: 1 to 1
: in the range of 60.
23. include the acid of less than 4 pKa according to the application described in claim 15, described application.
24. according to the application described in claim 23, and wherein said acid is the acid in the following: phosphoric acid, phosphonic acids, high iodine
Acid, selenic acid, maleic acid, oxalic acid, dichloroacetic acid and secondary amino three (methylene) tri methylene phosphonic acid, and wherein said acid is with 4%-20%
(w/v) concentration exists.
25. form sediment selected from cyclodextrin or cyclisation straight chain according to the application described in claim 15, one or more dextrin wherein said
Powder.
26. according to the application described in claim 15, and wherein said reagent comprises one or more alcohol, wherein said one or many
Plant alcohol and be selected from ethanol, methanol and propanol, and wherein said determining alcohol is 0.1%-10%v/v.
27. comprise detergent according to the application described in claim 15, wherein said reagent.
28. provide with many part system according to the application described in claim 15, wherein said reagent.
29. 1 kinds of reagent, comprise:
(a) protein-complexing dye,
(b) one or more dextrin, and
(c) one or more alcohol,
Described reagent is suitable for when protein detects in the presence of detergent or surfactant and/or quantifies method of protein
Middle use, and the concentration of one or more dextrin wherein said is 1-100mg/ml.
30. is 0.1%-10%v/v according to the reagent described in claim 29, wherein said determining alcohol.
31. 1 kinds of reagent, described reagent comprises:
(a) protein-complexing dye, and,
(b) one or more dextrin,
Described reagent is suitable for when protein detects in the presence of detergent or surfactant and/or quantifies method of protein
Middle use, and wherein, in providing the reagent for using, described protein composite dye is with 0.001%-0.1% (w/v)
Concentration exist, and the concentration of one or more dextrin wherein said is 1-100mg/ml.
32. according to the reagent described in claim 31, and wherein said protein-complexing dye is with 0.005%-0.5%'s (w/v)
Concentration exists.
33. 1 kinds of reagent, described reagent comprises:
(a) protein composite dye,
(b) one or more dextrin, and
C () has the acid of 4 or lower pKa,
Described reagent is suitable for when protein detects in the presence of detergent or surfactant and/or quantifies method of protein
Middle use, and wherein, in providing the reagent for using, described acid exists with the concentration of 4% to 20% (w/v), and
The concentration of one or more dextrin wherein said is 1-100mg/ml.
34. according to the reagent according to any one of claim 29-33, wherein, in use, dilutes described reagent, and tries
The ratio of agent and diluent is in the range of 1: 1 to 1: 60.
35. have the pKa in the range of 1 to 3 according to the reagent described in claim 33, wherein said acid.
36. according to the reagent according to any one of claim 29-33 and 35, and wherein said reagent comprises acid, wherein said acid
Selected from phosphoric acid, phosphonic acids, periodic acid, selenic acid, maleic acid, oxalic acid, dichloroacetic acid and secondary amino three (methylene) tri methylene phosphonic acid.
37. according to the reagent according to any one of claim 29-33 and 35, and wherein said acid is dense with 4%-20%'s (w/v)
Degree exists.
38. according to the reagent according to any one of claim 29-33 and 35, wherein
Described reagent also comprises solubilizing agent.
39. according to the reagent according to any one of claim 29-33 and 35,
Wherein said dyestuff is Coomassie dye.
40. are in 1-10mg/ml according to the reagent according to any one of claim 29-33 and 35, wherein said dextrin concentration
In the range of.
41. according to the reagent according to any one of claim 29-33 and 35,
Wherein said reagent comprises one or more alcohol, and one or more alcohol wherein said is selected from ethanol, methanol and propanol.
42. according to the reagent described in claim 41, and one or more alcohol wherein said is selected from ethanol, methanol and propanol, and
Wherein said determining alcohol is 0.1%-10%v/v.
43. provide with two parts according to the reagent according to any one of claim 29-33 and 35, wherein said reagent.
44. comprise dyestuff and optional alcohol according to the reagent described in claim 43, a part in wherein said two parts
And/or acid, and another part comprises one or more dextrin.
45. provide with aqueous components according to the reagent described in claim 43, wherein said two parts.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0608377A GB2437545B (en) | 2006-04-28 | 2006-04-28 | Dextrin-containing reagents for detecting and quantifying proteins |
| GB0608377.8 | 2006-04-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101473228A CN101473228A (en) | 2009-07-01 |
| CN101473228B true CN101473228B (en) | 2016-11-30 |
Family
ID=
Non-Patent Citations (3)
| Title |
|---|
| Dulley J R et al..A simple technique for eliminating interference by detergents in the lowry method of protein determination.《Analytical Biochemistry》.1975,第64卷(第1期),136-141. * |
| Luo Xi-Yaun et al..Detergent interference with lowry assay of bovine milk fat globule membrane protein.《Biochemical Archives》.1997,第13卷(第4期),319-326. * |
| Pei-Pei Xu et al.Interference by cyclodextrins in protein determination by the Bradford method.《Microchemical Journal》.1994,第49卷85-90. * |
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