EP1334353A1 - Procede de separation et de detection de proteines par electrophorese - Google Patents
Procede de separation et de detection de proteines par electrophoreseInfo
- Publication number
- EP1334353A1 EP1334353A1 EP01996736A EP01996736A EP1334353A1 EP 1334353 A1 EP1334353 A1 EP 1334353A1 EP 01996736 A EP01996736 A EP 01996736A EP 01996736 A EP01996736 A EP 01996736A EP 1334353 A1 EP1334353 A1 EP 1334353A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- proteins
- separation step
- separation
- protein
- electrophoresis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 118
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 118
- 238000000034 method Methods 0.000 title claims abstract description 66
- 238000001962 electrophoresis Methods 0.000 title description 13
- 238000000926 separation method Methods 0.000 claims abstract description 57
- 238000005251 capillar electrophoresis Methods 0.000 claims abstract description 17
- 238000001155 isoelectric focusing Methods 0.000 claims abstract description 16
- 238000001997 free-flow electrophoresis Methods 0.000 claims abstract description 14
- 238000002218 isotachophoresis Methods 0.000 claims abstract description 8
- 239000003550 marker Substances 0.000 claims abstract description 6
- 230000001413 cellular effect Effects 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 4
- 239000007850 fluorescent dye Substances 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 5
- 230000002209 hydrophobic effect Effects 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 229920002401 polyacrylamide Polymers 0.000 claims description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 150000002540 isothiocyanates Chemical class 0.000 claims description 3
- 238000001499 laser induced fluorescence spectroscopy Methods 0.000 claims description 3
- 150000007942 carboxylates Chemical class 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 150000003573 thiols Chemical class 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 claims 20
- 229920000936 Agarose Polymers 0.000 claims 1
- ZCSCIKKXDSLFDO-UHFFFAOYSA-N N-(2,5-dioxopyrrol-3-yl)-2-iodoacetamide Chemical compound ICC(=O)NC=1C(=O)NC(C1)=O ZCSCIKKXDSLFDO-UHFFFAOYSA-N 0.000 claims 1
- 125000005997 bromomethyl group Chemical group 0.000 claims 1
- 235000018417 cysteine Nutrition 0.000 claims 1
- 150000001945 cysteines Chemical class 0.000 claims 1
- 235000018977 lysine Nutrition 0.000 claims 1
- 150000002669 lysines Chemical class 0.000 claims 1
- 229920001059 synthetic polymer Polymers 0.000 claims 1
- 239000000499 gel Substances 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 108010026552 Proteome Proteins 0.000 description 8
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 8
- 239000000975 dye Substances 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000005684 electric field Effects 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 239000003792 electrolyte Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000010979 ruby Substances 0.000 description 4
- 229910001750 ruby Inorganic materials 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000001917 fluorescence detection Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 238000001712 DNA sequencing Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241000238413 Octopus Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000959 ampholyte mixture Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- -1 for example Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- 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
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
-
- 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
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis
Definitions
- the invention relates to a method for separating and detecting proteins in order to accelerate proteome analysis.
- a proteome is the term used to describe and quantify all proteins of an organism, a cell, an organelle or a body fluid at a defined point in time and under precisely defined conditions.
- proteins are examined to determine which proteins play which role in biological processes and which proteins are of particular importance in interaction with other proteins.
- the question of the extent to which chemicals, active substances and other external factors (environmental influences, heat, cold, lack of water, pH value, etc.) influence cellular protein expression is investigated in the context of proteome analysis.
- proteome analysis also tries to find out which proteins in which protein constellations can be responsible for which side effects.
- the question is examined whether the protein expression of microorganisms can be influenced in such a way that the space-time yields of fermentative production processes can be improved.
- sample preparation poses a significant problem.
- the course is set to separate and identify even complex protein patterns. It has been found that the solubility has a significant influence on the separation of proteins. Proteins that dissolve quickly in water generally do not pose any problems with regard to separability.
- Proteins with stable secondary or tertiary structures that are sparingly water-soluble are stabilized in terms of their solubility behavior by adding chaotropic substances such as guanidine hydrochloride or urea.
- chaotropic substances such as guanidine hydrochloride or urea.
- undesired reactions of individual proteins can occur, whereby a protein originally present in one form changes into several forms and thus further increases the heterogeneity of the sample already present.
- Membrane proteins the natural environment of which are lipid membranes and which tend to agglomerate and become insoluble again when they are isolated from one another, are particularly difficult to handle. These hydrophobic proteins can only be kept in the dissolved state if detergents are added, which, however, can frequently impair the subsequent stages of protein separation.
- High protein concentrations are highly desirable for most separation and evaluation procedures, but are associated with the risk that aggregations will form.
- low protein concentrations which have a positive effect on the solubility behavior, are associated with additional preparation steps before the actual separation.
- Proteins have the character of zwitterions and can therefore be positively or negatively charged. Using electrophoresis methods, individual components can be separated according to their mobility in the electrical field. The electrophoretic
- Mobility of each protein is a characteristic value.
- proteome analysis two electrophoresis methods are used, isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- SDS sodium dodecyl sulfate
- This method provides for the separation of the proteins in the first dimension with the isoelectric focusing according to their isoelectric point.
- the second dimension is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the proteins according to their size.
- This method is currently the only procedure with which complex protein mixtures can be separated with high resolution.
- the advantages of the 2D-PAGE process are the two mutually complementary separation principles, namely IEF and SDS-PAGE processes according to load and molecular weight. In principle, this technique can be used with all proteins.
- the resolution of this method can be increased significantly by using narrow pH gradients in isoelectric focusing ("zoom gels").
- Staining is carried out, for example, with dyes such as Coomassie blue, with colloidal silver, with zinc / imidazole or with fluorescent dyes such as Sypro ® Ruby, Sypro ® Orange or Sypro ® Red. All dyeing methods used have in common that the bond between protein and dye reagent is not covalent , but on the basis of ionic, hydrophobic or Van der Waal interactions. After staining, the gels are usually digitized using a scanner or a fluorescence scanner.
- US 6,043,025 and US 6,127,134 describe a process and a kit with which one can detect differences in two or more protein samples.
- the protein extracts from different samples are covalently labeled with different, positively charged fluorescent dyes, combined and subjected to 2D PAGE. Identical proteins from the different samples can then be detected and quantified on the basis of their different fluorescence wavelengths in the same gel.
- the proteins of a first cell are prepared using known treatment techniques, the first cell originating from a first group of cells.
- the proteins are covalently labeled with a first dye from a pair of dyes.
- a second cell is prepared using known treatment techniques that were taken from a second group of cells.
- the proteins of this second cell are covalently labeled with a second dye.
- radioactive methods can also be used.
- the cells are mixed with certain isotope-labeled compounds such as 35 S-cysteine or 32 PO 4 3 " .
- a phosphor imager is usually used for digitization in these cases.
- image evaluation programs such as MELANIE, PDQUEST, IMAGEMASTER or The protein spots are then detected, quantified and assigned in the digitized gels obtained.
- the entire procedure from electrophoresis to staining, detection and quantification according to the 2D-PAGE method is very complex.
- the object of the invention is to develop a separation method for proteins which reduces the use of gels, is quick and easy to carry out and can be automated, and allows simple quantification of the separated proteins.
- this object is achieved by a method for the separation and detection of proteins, the protein samples being contained in a separation buffer solution and the following method steps being carried out:
- the protein samples are broken down into individual fractions according to free-flow electrophoresis with isoelectric focusing (IEF) or isotachophoresis and with a
- the protein fractions are separated in one or more capillaries according to capillary electrophoresis, at least one labeling substance being detected in the individual capillary (s).
- protein fractions obtained after the first separation step are linked with a reactive fluorescent dye.
- a reactive fluorescent dye This can be done, for example, by coupling the N-hydroxysuccinimide esters (NHS esters) or isothiocyanates of fluorescent dyes to free amino groups of the proteins. Free amino groups are preferably the N-termini or lysine selected as coupling sites.
- the dyes can be covalently bound to the individual proteins in the process proposed according to the invention.
- protein fractions obtained after the first separation step are mixed with a marking substance.
- a marking substance e.g. Sypro® Ruby, Sypro Orange or Sypro Red.
- the fluorescent dyes Sypro Ruby, Sypro Orange or Sypro®Red can be bound by adsorption, e.g. by hydrophobic, ionic or Van der Waal's forces.
- capillaries In a second separation step, capillaries can be used which are either provided with a polyacrylamide gel or which do not contain this substance, i.e. are empty.
- the individual proteins are detected in the second separation step using laser-induced fluorescence. Sensitive fluorescence detection ensures a wide dynamic range and high sensitivity.
- sodium dodecyl sulfate can be added to the separation buffer. Due to the high separation performance of capillary electrophoresis, a significantly better resolution in the second dimension is achieved compared to the 2D-PAGE gel process; Furthermore, the high degree of automation of both separation steps, the FFE and the capillary electrophoresis, enables a significantly higher throughput and thus a better statistical validation of the results obtained. In order to ensure the parallel processing of a higher number of protein samples after free-flow electrophoresis, all wells of a microtiter plate can be detected and quantified separately in parallel by as many capillaries. This can be achieved, for example, by using a commercially available device for DNA sequencing.
- several different fluorescent dyes can be detected simultaneously in each capillary in the second separation step.
- the sensitive fluorescence detection ensures a large dynamic range and high sensitivity. Due to the high separation performance of capillary electrophoresis, a significantly better resolution in the second dimension is achieved compared to the 2D PAGE gel process. Due to the high degree of automation of both separation steps, FFE and capillary electrophoresis, a significantly higher throughput and thus a better statistical validation of the measured results is expected.
- the separation of the protein samples in the first separation step can e.g. in an advantageous manner in a microtiter plate, a microtiter plate being used, the number of wells of which corresponds to that of the separated protein fractions.
- the number of capillaries used in the second separation step according to capillary electrophoresis advantageously corresponds to the number of sample fractions introduced into the microtiter plate.
- two different free-flow electrophoresis methods can be used for the method proposed according to the invention: isoelectric focusing and isotachophoresis.
- isotachophoresis can be used in the first separation step.
- a potential gradient is formed in the electrical field in a discontinuous buffer system consisting of the lead and secondary electrolytes.
- the field strength is higher in the area of ions with low mobility than in the area of more mobile ions. Since all ions must migrate at the same speed, pure zones of the individual proteins are formed from the protein sample mixture. In the equilibrium state, the ion with the highest mobility follows the lead ion of the lead electrolyte, the one with the lowest mobility moves in front of the follow-up electrolyte, the others move in between in the order of decreasing mobility.
- interval isotachophoresis is performed (Gerhard Weber and Petr Bocek in Electrophoresis 1998, 19, 3090 - 3093). After the protein sample and the electrolytes have been applied to the free-flow electrophoresis chamber, high voltage is applied for 2 minutes to separate the proteins, then the separated fractions are conveyed without tension via a series of tubes into the individual wells of a microtiter plate.
- individual protein fractions are obtained in microtiter plates in this electrophoresis device.
- the proteins in these fractions can be linked both to a marker, for example a fluorescent dye such as Sypro®Orange, Sypro®Red or Sypro®Ruby, and to at least one reactive fluorescent dye.
- a marker for example a fluorescent dye such as Sypro®Orange, Sypro®Red or Sypro®Ruby
- Derivatives such as the N-hydroxysuccinimide esters (NHS esters) or the isothiocyanates of fluorescent dyes, which are coupled to free amino groups of the proteins, are suitable for this. N-termini or lysine of the proteins or the protein fractions are particularly suitable.
- the second separation step e.g. Separate covalently fluorescence-labeled proteins using capillary electrophoresis.
- the one or more capillary tubes used can be filled on the one hand with a polyacryamide gel, on the other hand the use of unloaded, i.e. empty capillary tube possible.
- Detection preferably follows with laser-induced fluorescence, and sodium dodecyl sulfate can be added to the separation buffer to improve the running behavior of the proteins in capillary electrophoresis.
- all wells of the microtiter plate are separately detected and quantified in parallel in as many capillaries.
- a commercially available device for DNA sequencing is used, for example a Mega-BACE from Amersham Pharmacia or another device of a similar design.
- An advantage over conventional 2D gel electrophoresis with subsequent non-covalent staining with image evaluation for quantification is that the electropherograms obtained by the method described here can be easily quantified using commercially available software.
- several different marking substances such as, for example, fluorescent dyes
- only a fluorescent dye or fluorescent substance can be detected just as well.
- the multiple samples analyzed in one run are proteins from different cells or from cells in different stages of development or from cells that were exposed to different external conditions (such as heat, cold, active substances, chemicals, etc.).
- Dissolution of the protein samples or the protein samples of cellular origin in the second Dimension compared to the 2D PAGE method can be achieved. Due to the significantly better automatability of both methods, free-flow electrophoresis and capillary electrophoresis, a significantly higher throughput and thus a better statistical validation of the results will take place.
- a program sequence for evaluating the electropherograms is to be created; furthermore, in order to implement the method proposed according to the invention, a free-flow electrophoresis apparatus (“Octopus” from Dr. Weber GmbH), for example, and, for example, a Mega-BACE sequencer from Amersham or a similar device are required.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE2000156838 DE10056838A1 (de) | 2000-11-16 | 2000-11-16 | Verfahren zur Trennung und Detektion von Proteinen |
DE10056838 | 2000-11-16 | ||
DE2001120803 DE10120803A1 (de) | 2001-04-27 | 2001-04-27 | Verfahren zur Trennung und Detektion von Proteinen |
DE10120803 | 2001-04-27 | ||
DE2001135497 DE10135497A1 (de) | 2001-07-20 | 2001-07-20 | Verfahren zur Trennung und Detektion von Proteinen |
DE10135497 | 2001-07-20 | ||
PCT/EP2001/013195 WO2002040983A1 (fr) | 2000-11-16 | 2001-11-14 | Procede de separation et de detection de proteines par electrophorese |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1334353A1 true EP1334353A1 (fr) | 2003-08-13 |
Family
ID=27214156
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01996736A Withdrawn EP1334353A1 (fr) | 2000-11-16 | 2001-11-14 | Procede de separation et de detection de proteines par electrophorese |
Country Status (7)
Country | Link |
---|---|
US (1) | US20040031683A1 (fr) |
EP (1) | EP1334353A1 (fr) |
JP (1) | JP2004514136A (fr) |
AU (1) | AU1702902A (fr) |
CA (1) | CA2428372A1 (fr) |
NO (1) | NO20032209L (fr) |
WO (1) | WO2002040983A1 (fr) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10047088C2 (de) * | 2000-09-21 | 2002-10-17 | Gerhard Weber | Medium für analytische und präparative Elektrophorese |
DE10063096C1 (de) * | 2000-12-18 | 2002-09-12 | Gerhard Weber | Elektrophoresevorrichtung, Elektrophoreseverfahren unter Verwendung einer Elektrophoresevorrichtung und Verwendung der Elektrophoresevorrichtung |
DE10063097B4 (de) * | 2000-12-18 | 2007-04-19 | Becton, Dickinson And Co. | Elektrophoresevorrichtung |
SE0202401D0 (sv) * | 2001-12-11 | 2002-08-13 | Thomas Laurell | Biomolecule handling method and machine using an arry dispenser |
US7850835B2 (en) * | 2003-05-09 | 2010-12-14 | Life Technologies Corporation | Solution phase electrophoresis device, components, and methods |
US7622028B2 (en) * | 2003-05-09 | 2009-11-24 | Life Technologies Corporation | Solution phase electrophoresis device, components, and methods |
DE102005020134A1 (de) * | 2005-04-29 | 2006-11-02 | Becton, Dickinson And Co. | Verfahren und Vorrichtung zur Durchführung eines parallelen und simultanen Mehrfachprozesses der trägerfreien isoelektrischen Fokussierung |
US8562804B2 (en) | 2006-07-20 | 2013-10-22 | The Board Of Trustees Of The Leland Stanford Junior University | Fluorescent finger prints for indirect detection in isotachophoresis |
EP2059799B1 (fr) * | 2006-08-29 | 2017-07-12 | Becton Dickinson and Company | Procédé et dispositif d'électrophorèse libre sans vecteur |
US20080220442A1 (en) * | 2006-12-06 | 2008-09-11 | Proteinics | Difference detection methods using isoelectric focusing chips |
EP2623975B1 (fr) | 2006-12-26 | 2016-02-03 | Sekisui Chemical Co., Ltd. | Procédé de mesure du taux d'hémoglobine stable A1c et de glucose |
JP4854503B2 (ja) * | 2006-12-27 | 2012-01-18 | 積水化学工業株式会社 | 安定型ヘモグロビンA1cの測定方法 |
DE602007011603D1 (de) * | 2007-08-29 | 2011-02-10 | Agilent Technologies Inc | On-Chip-Analyse kovalent markierter Probenspezies |
US8614059B2 (en) * | 2007-12-14 | 2013-12-24 | The Johns Hopkins University | Purification and concentration of proteins and DNA from a complex sample using isotachophoresis and a device to perform the purification |
US8865401B2 (en) | 2007-12-14 | 2014-10-21 | The Johns Hopkins University | Purification and concentration of proteins and DNA from a complex sample using isotachophoresis and a device to perform the purification |
US8846314B2 (en) * | 2009-03-03 | 2014-09-30 | The Board Of Trustees Of The Leland Stanford Junior University | Isotachophoretic focusing of nucleic acids |
JP5513802B2 (ja) * | 2009-08-04 | 2014-06-04 | ホーユー株式会社 | 等電点電気泳動用ゲル及び等電点電気泳動方法 |
US8721858B2 (en) * | 2010-03-12 | 2014-05-13 | The Board Of Trustees Of The Leland Stanford Junior University | Non-focusing tracers for indirect detection in electrophoretic displacement techniques |
US8986529B2 (en) | 2010-09-13 | 2015-03-24 | The Board Of Trustees Of The Leland Stanford Junior University | Isotachophoresis having interacting anionic and cationic shock waves |
US8524061B2 (en) | 2010-11-29 | 2013-09-03 | The Board Of Trustees Of The Leland Stanford Junior University | On-chip hybridization coupled with ITP based purification for fast sequence specific identification |
CN102135524B (zh) * | 2010-12-15 | 2013-08-07 | 中国水产科学研究院黄海水产研究所 | 高温环境下海水鱼类体表粘液功能蛋白检测方法 |
WO2017132630A1 (fr) | 2016-01-29 | 2017-08-03 | Purigen Biosystems, Inc. | Isotachophorèse destinée à la purification d'acides nucléiques |
CN115569515A (zh) | 2017-08-02 | 2023-01-06 | 普瑞珍生物系统公司 | 用于等速电泳的系统、设备和方法 |
CN113189182B (zh) * | 2021-04-25 | 2023-05-23 | 湖南省作物研究所 | 一种水溶蛋白鉴定甘蓝型油菜杂交种种子纯度方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5468359A (en) * | 1988-11-14 | 1995-11-21 | Anthony R. Torres | Method of determining presence of an analyte by isoelectric focusing |
US6127134A (en) * | 1995-04-20 | 2000-10-03 | Carnegie Mellon University | Difference gel electrophoresis using matched multiple dyes |
US6277259B1 (en) * | 1998-04-24 | 2001-08-21 | Enterprise Partners Ii | High performance multidimensional proteome analyzer |
US6764817B1 (en) * | 1999-04-20 | 2004-07-20 | Target Discovery, Inc. | Methods for conducting metabolic analyses |
DE19932270A1 (de) * | 1999-07-05 | 2001-01-11 | Univ Schiller Jena | Verfahren zur mehrdimensionalen Analyse eines Proteoms |
-
2001
- 2001-11-14 AU AU1702902A patent/AU1702902A/xx not_active Withdrawn
- 2001-11-14 CA CA002428372A patent/CA2428372A1/fr not_active Abandoned
- 2001-11-14 WO PCT/EP2001/013195 patent/WO2002040983A1/fr active Application Filing
- 2001-11-14 JP JP2002542861A patent/JP2004514136A/ja active Pending
- 2001-11-14 EP EP01996736A patent/EP1334353A1/fr not_active Withdrawn
- 2001-11-15 US US10/416,460 patent/US20040031683A1/en not_active Abandoned
-
2003
- 2003-05-15 NO NO20032209A patent/NO20032209L/no not_active Application Discontinuation
Non-Patent Citations (4)
Title |
---|
OH-ISHI M; SATOH M; MAEDA T: "PREPARATIVE TWO-DIMENSIONAL GEL ELECTROPHORESIS WITH AGAROSE GELS IN THE FIRST DIMENSION FOR HIGH MOLECULAR MASS PROTEINS", ELECTROPHORESIS, vol. 21, no. 9, May 2000 (2000-05-01), WEINHEIM, DE, pages 1653 - 1669, XP001056980 * |
See also references of WO0240983A1 * |
WEBER G; BOCEK P: "Interval isotachophoresis for purification and isolation of ionogenic species", ELECTROPHORESIS, vol. 19, no. 18, WEINHEIM, DE, pages 3090 - 3093, XP007900331, DOI: doi:10.1002/elps.1150191808 * |
WEBER G; BOCEK P: "RECENT DEVELOPMENTS IN PREPARATIVE FREE FLOW ISOELECTRIC FOCUSING", ELECTROPHORESIS, vol. 19, no. 10, July 1998 (1998-07-01), WEINHEIM, DE, pages 1649 - 1653, XP001057209 * |
Also Published As
Publication number | Publication date |
---|---|
CA2428372A1 (fr) | 2002-05-23 |
JP2004514136A (ja) | 2004-05-13 |
WO2002040983A1 (fr) | 2002-05-23 |
US20040031683A1 (en) | 2004-02-19 |
NO20032209L (no) | 2003-07-15 |
AU1702902A (en) | 2002-05-27 |
NO20032209D0 (no) | 2003-05-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1334353A1 (fr) | Procede de separation et de detection de proteines par electrophorese | |
DE60110769T2 (de) | Verfahren und vorrichtung zur analyse von geladenen molekülen durch isoelektrische fokussierung einer lösung | |
DE60112276T2 (de) | Elektrophoretische trennung von verbindungen | |
DE60222427T2 (de) | Puffer-arrays zum nachweis von biomolekülen anhand ihres isoelektrischen punktes | |
DE2828179C2 (fr) | ||
DE69115690T2 (de) | Verfahren und gerät zum folgerichtigen chemischen reaktionsablauf | |
DE3586154T3 (de) | Verbessertes verfahren zur immunofixierungselektrophorese. | |
DE10047088C2 (de) | Medium für analytische und präparative Elektrophorese | |
Chee et al. | Reproducible and high-speed separation of basic drugs by capillary zone electrophoresis | |
Zhang et al. | Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse | |
Pérez‐Ruiz et al. | Development and validation of a capillary electrophoresis method with laser‐induced fluorescence detection for the determination of captopril in human urine and pharmaceutical preparations | |
DE60308716T2 (de) | Verfahren zur isoelektrischen auftrennung mittels ph-wert-voreinstellung | |
EP2118666A1 (fr) | Procédé pour normaliser la concentration d'analytes dans un échantillon d'urine | |
DE69101279T2 (de) | Neutrale oder positiv geladene Farbstoffe für Lösungen zum Laden von elektrophoretischen Proben. | |
DE602004005843T2 (de) | Vorkonzentrierende Verbindungstelle für die Kupplung von flüssiger Chromatographie und Kapillarelektrophorese | |
DE2603069A1 (de) | Verfahren und vorrichtung zur bestimmung des gesamtproteingehaltes oder einzelner aminosaeuren | |
DE112005003114T5 (de) | Verfahren zum Trennen von Protein, Verfahren zum Färben von Protein und flüssiges Proteinfärbemittel sowie Proteinfärbekit zur Verwendung bei diesen Verfahren | |
DE69007773T2 (de) | Elektrophoretische Trennung in einem gelfreien Medium mittels gelösten Polymeren. | |
EP0693559A1 (fr) | Procédé pour la détermination quantitative des protéines glycés | |
DE10120803A1 (de) | Verfahren zur Trennung und Detektion von Proteinen | |
DE10135497A1 (de) | Verfahren zur Trennung und Detektion von Proteinen | |
EP2918597A1 (fr) | Procédé et kit de surveillance de préparation d'échantillons, de réactions de marquage chimique et de réactions d'anticorps | |
DE2529606A1 (de) | Isoelektrische fokusierung | |
DE10056838A1 (de) | Verfahren zur Trennung und Detektion von Proteinen | |
DE1598105B1 (de) | Matrixsubstanz fuer die Elektrophorese |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20030508 |
|
AK | Designated contracting states |
Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR |
|
AX | Request for extension of the european patent |
Extension state: AL LT LV MK RO SI |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: EIPEL, HEINZ Inventor name: WEBER, GERHARD Inventor name: PLATSCH, HERBERT Inventor name: HAMMERMANN, MARKUS |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: DR. WEBER GMBH Owner name: BASF SE |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20080603 |