WO1995019339A1 - Agents de solubilisation de biomolecules constitues par des zwitterions non detergents et leurs utilisations - Google Patents
Agents de solubilisation de biomolecules constitues par des zwitterions non detergents et leurs utilisations Download PDFInfo
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- WO1995019339A1 WO1995019339A1 PCT/FR1995/000035 FR9500035W WO9519339A1 WO 1995019339 A1 WO1995019339 A1 WO 1995019339A1 FR 9500035 W FR9500035 W FR 9500035W WO 9519339 A1 WO9519339 A1 WO 9519339A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
- C07D213/16—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom containing only one pyridine ring
- C07D213/20—Quaternary compounds thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
- C07C309/01—Sulfonic acids
- C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
- C07C309/03—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
- C07C309/13—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton
- C07C309/14—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton containing amino groups bound to the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
- C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
Definitions
- the present invention relates to new biomolecule solubilizing agents such as proteins, which can be used to solubilize or maintain in solution biomolecules during processes such as the methods of purification, separation and analysis of biomolecules, for example by techniques electrophoresis or chromatography.
- solubilizers are detergents, salts and urea, but they all have drawbacks.
- detergents are described for example by Navarrete et al in Biochimica and Biophysica Acta, 728, 1983, p. 403-408.
- the detergents mentioned by these authors such as sodium do ⁇ ecylsufate, Triton X-100, octylglucosides and zwittergents are effective because they have for the most part a strong power of solubilization of proteins but this is generally accompanied by a strong denaturing effect due to the destruction of hydrophobic type bonds which lead to the aggregation of proteins between them but are also responsible for the conformation of proteins, necessary for their biological properties.
- detergents in addition to their high price, detergents have the disadvantage of forming micelles which are often of large diameter, which limits their use in cases where dialysis processes, gels and certain columns are used. chromatography.
- the salts can be used as solubilizing agents for proteins, when their aggregation is due to the formation of ionic bonds between protein molecules.
- the use of the salts is impossible in all cases where an increase in the conductivity of the solution is detrimental, for example in electrophoretic processes and ion exchange chromatography processes.
- Urea can also be used as a protein solubilizing agent because it has a strong chaotropic power, and ensures the breaking of hydrogen-type bonds and those involving dipoles.
- urea has two major drawbacks which are respectively its denaturing effect, and the risks of carbamylation of free amines of proteins by the cyanate groups formed by the spontaneous decomposition of urea.
- biomolecule solubilizing agents having in particular the following properties: - not to cause denaturation of the biomolecules by modification of their conformation,
- the present invention specifically relates to new agents for solubilizing biomolecules such as proteins, which have these advantageous properties.
- the agent for solubilizing biomolecules consists of a non-detergent zwitterion corresponding to one of the following formulas: RI
- -B represents a saturated hydrocarbon chain of 1 to 6 carbon atoms optionally comprising in its chain one or more oxygen and / or sulfur atoms, which can additionally comprise one or more substituents constituted by OH groups, - R-, R2 and R ⁇ , which may be identical or different, represent hydrocarbon, aliphatic, linear or branched, cyclic or aromatic groups, having at most 7 carbon atoms, comprising or not one or more groups -0-, -S- and / or -OH, R 1 and R 2 can also together form a saturated hydrocarbon chain of 4 to 8 carbon atoms, optionally comprising one or more OH substituents and / or one or more oxygen atoms and / or sulfur,
- R represents with X an unsaturated heterocycle with one or more rings containing at most 9 carbon atoms and one or more heteroatoes chosen from 0, S, N and P, and optionally comprising one or more OH substituents, and
- R 1 , R 2 and / or R 3 represent a linear aliphatic group, the latter preferably has at most 5 carbon atoms.
- the zwitterions used in the invention are different from the detergent zwitterions described by Navarrete et al in Biochimica and Biophysica Acta, 728, 1983, p. 403-408.
- the non-detergent zwitterions of the invention do not have long hydrophobic chains; therefore, they have a very weak denaturing effect and they do not form micelles which are difficult to remove.
- they can be easily eliminated at the end of the operation to give pure biomolecules and they do not interfere not with the ionic strength of the solutions.
- they are easy to synthesize and inexpensive.
- Methacrylamide derivatives zwitterions were also used to make gels electrophoresis on 1 sulfobetaine groups, for the separation of proteins, as described by Hjelmeland et al in Electrophoresis 1981, 2, p. 82-90. In this case, on the one hand, these are not zwitterions of the same type as those of the invention, and on the other hand, they do not have the function of solubilizing biomolecules.
- B can represent a saturated hydrocarbon chain, having from 1 to 6 carbon atoms, optionally comprising in its chain one or more oxygen and / or sulfur atoms and optionally a or more substituents consisting of OH groups.
- B represents the group - (CH 2 ) ⁇ ⁇ with 1 being equal to 3 or 4.
- the aliphatic, cyclic or aromatic hydrocarbon groups used for R 1 , R 2 and R 3 can be in particular alkoxyalkyl, alkylthioalkyl, linear or branched alkyl groups of 1 to 7 carbon atoms, cycloalkyl groups of 5 to 7 carbon atoms and aryl groups of 5 to 7 carbon atoms. All of these groups can additionally contain one or more OH substituents.
- R 1 and R 2 when R 1 and R 2 together form a saturated hydrocarbon chain optionally comprising one or more OH substituents and / or one or more oxygen and / or sulfur atoms in the chain, R 1 and R 2 form with X a heterocycle which may include other heteroatoms. Mention may be made, as examples of such heterocycles, of the piperidine, piperazine and morpholine nuclei.
- R 3 is preferably an alkyl group, for example the methyl group.
- R forms with X an unsaturated heterocycle with one or more nuclei
- R may be, for example, the pyridine, oxazole, thiazole, benzothiazole or benzoxazole nucleus.
- non-detergent zwitterions used in the invention can be prepared by conventional methods, starting from sultones, lactones, ⁇ -chloroacids or corresponding chloroalcohols by reaction with the amines or phosphines of formula:
- a ra-chloroacid In the case where Y represents C00 ⁇ , a ra-chloroacid, a lactone or acrylic acid can be reacted with the corresponding amine or phosphine.
- biomolecule solubilizers of the invention can be used in various methods involving biomolecules such as the methods of solubilization, extraction and purification of biomolecules, and the methods of separation of biomolecules by electrophoresis or chromatography techniques, for example separations by isoelectrofocusing, chromatofocusing and by ion exchange chromatography.
- solubilizing agents can be used alone or as a mixture, and optionally in combination with other known solubilizing agents such as weakly denaturing detergents, for example the product marketed under the name TRITON X 100 or (tert-octylphenyl- (oligoethylene) oxide), octyl glucopyranoside and 3 [3-cholamidopropyl] dimethylamonio propane sulfonate.
- the solubilizers of the invention can also be used for the renaturation of certain biomolecules such as enzymes, after these have been denatured, for example by a highly denaturing solubilizing agent
- solubilizers When used in methods of separation or purification of biomolecules by electrophoresis, they can be added to the solution of biomolecules to be separated or to be purified, and / or to the separation medium.
- they can be added to the preparation solution and / or to the gel rehydration solution.
- they can be added to the conductive solutions.
- solubilizing agents are used in separation or purification processes by chromatography, they can be added to the solution of biomolecules to be separated or to be purified and / or to the conditioning and / or elution solutions.
- solubilizing agents are used for the renaturation of biomolecules denatured, they can be added to the solutions used in the renaturation process.
- biomolecules to which the solubilizing agents of the invention apply can consist of any biological molecule, for example nucleic acids, carbohydrates, lipids, coenzymes and in particular proteins.
- Proteins can consist in particular of enzymes, plasma proteins, nuclear proteins, membrane proteins, antibodies and structural proteins.
- FIG. 1 is a diagram representing the efficiency of extraction of microsomal proteins obtained in Examples 9 to 15.
- FIG. 2 is a diagram representing the yields of maintenance in solution of nuclear proteins in dialysis solutions after saline extraction.
- FIG. 3 is a photograph illustrating the results obtained during one-dimensional gel electrophoresis (isoelectrofocusing) of a protein in the presence of a solubilizing agent of
- FIGS. 4 and 5 are photographs illustrating the results obtained during the separation by two-dimensional gel electrophoresis of a complex mixture of proteins in the presence and in the absence of a solubilizing agent according to the invention.
- FIG. 6 is a diagram illustrating the enzymatic activity of ⁇ -galactosidase in the presence of various solubilizing agents in accordance with the invention.
- FIG. 7 is a diagram illustrating the enzymatic activity of ⁇ -galactosidase as a function of the concentration of compound No. 2 of the dilution medium.
- FIG. 8 is a diagram illustrating the percentage of enzymatic activity recovered in the presence of compound No. 2 after denaturation of ⁇ -galactosidase by urea.
- FIG. 9 is a photograph illustrating the results of analysis, by gel electrophoresis of the fractions originating from a chromatography column in the presence and in the absence of compound No. 2.
- Example 2 Preparation of pyridinium propane sulfonate (compound No. 2).
- the commercial product purchased from Fluka was then purified by passage in aqueous solution over mixed ion exchange resins.
- P3-X63-Ag8 mouse secreting plasmacytoma cells are prepared by culture and the proteins are labeled by incorporation of sulfur-labeled amino acids 35.
- the cells are recovered, washed in saline and lysed by lysis osmotic for 30 min, at 0 ° C. in the following solution: hydroxyethyl piperazine ethane sulfonic acid (HEPES): at lmmol / l,
- the extraction is carried out at 20 ° C.
- the extraction is carried out in all cases for 1 h at 0 ° C., then the non-solubilized proteins are removed by centrifugation (200,000 g, 1 h ).
- the supernatant liquids which contain the solubilized proteins are recovered.
- the extraction yield is calculated by comparing the amounts of protein solubilized by the solutions of examples 10 to 15 to that solubilized by the solution of example 9 taken as extraction reference, since it contains a very effective solubilizing agent but highly denaturing, sodium dodecyl sulfate (SDS).
- SDS sodium dodecyl sulfate
- Examples 16 to 18 maintenance in solution of nuclear proteins after saline extraction.
- BASC 6-C2 urine lymphoma cells are cultured and their proteins are labeled as before by incorporation of amino acids with 35 S sulfur. After washing in saline, the cells are lysed (20 min, 0 ° C.) by 20 volumes of the solution having the following composition:
- the nuclei are collected by centrifugation at low speed (1000g, 5min) and washed by resuspension in the solution having the composition given above and recentrifugation. This operation is repeated twice.
- the nuclear proteins are then extracted for 1 h at 0 ° C. with the following solution:
- the nucleic acids and the non-solubilized proteins are removed after extraction, by centrifugation (200,000 g, 1 h).
- the solubilized proteins are then dialyzed against the solutions having the compositions given in table 3, using in each case a volume of solution equal to 100 times the volume of the solubilized protein extract.
- the yield of the protein solution is determined by comparing the amount of protein in solution obtained after dialysis to that obtained before dialysis. The results obtained, expressed in% of the quantity of proteins dissolved before dialysis, are given in Figure 2 attached.
- Examples 19 to 21 maintenance in solution of nuclear proteins after saline extraction.
- the same procedure is followed as in Examples 16 to 18, but a solution having the following composition is used for the extraction of nuclear proteins:
- solubilized proteins After elimination of the proteins and of the non-solubilized nucleic acids, by centrifugation under the same conditions as those described in Examples 16 to 18, the solubilized proteins are dialyzed against the solutions having the compositions given in Table 3 using in each case a volume of solution corresponding to 100 times the volume of extract. The results obtained, expressed as in the case of Examples 16 to 18 in% of the amount of proteins solubilized before dialysis, are given in FIG. 2.
- Example 22 Separation of a protein by isoelectric focusing on gel.
- proteins are brought to their isoelectric point by the action of an electric field on a pH gradient.
- isoelectric focusing presents a daunting problem of solubility because the proteins are brought to their isoelectric point where their solubility is minimal, in the absence of salts, which further contributes to increasing precipitation.
- isoelectric focusing is carried out in the presence of 8 mol / l of urea (highly denaturing condition).
- urea highly denaturing condition
- Isoelectrophoretic separation is carried out on a pH gradient using immobilized gels (Immobilines II from Pharmacia).
- the protein sample to be separated which, in this case, consists of a protein fragment of
- 110 kDa of fibronectin is loaded on the surface of the gel and the focusing is carried out with a field of 3V / cm for 3h, lOV / cm for 3h, lOOV / c for 18h and 200V / cm for lh.
- a one-dimensional gel is used which is stained with Coomassie blue as described by Neuhoff et al in Electrophoresis, 1988, 9, p. 255-262.
- FIG. 3 the migration tracks of the fibronectin HOkDa fragment protein are shown in the absence and in the presence of compound No. 2 in a linear gradient going from a pH of 7 to a pH of 4.
- a precipitation of the protein is observed before it reaches its isoelectric point, which results in a drag.
- the bottom track which corresponds to the migration of the protein in the presence of 1 mol / 1 of compound No. 2, we see that the protein migrates to its isoelectric point and we observe the separation of the isoforms into bands sharp, which shows that the protein remains soluble under these conditions.
- Example 23 Separation of plasma proteins.
- Example 22 The same procedure is followed as in Example 22 to separate plasma proteins by isoelectric focusing, either on a linear gradient from pH 3.5 to pH 5, adding or not adding 1 mol / 1 gels to the rehydration solution.
- compound # 2 In all cases, two-dimensional gels are used, and the migration is followed by an incubation of the strips for 10 mm in a solution having the following composition: - urea: 6 mol / 1,
- the transfer to polyacrylamide gel is then carried out by sealing the isoelectric focusing gel with 1% agarose in a 0.2mol / l solution of Bis-tris, 0.1 lmol / l of HCl, 0.2% of sodium dodecyl sulfate and 0.002% bromophenol blue.
- the proteins are separated by electrophoresis (40 mA by 160 mm X 200 mm X 1.5 mm gel) and then stained with Coomassie blue as in Example 22.
- Figures 4 and 5 illustrate the results obtained during the migration from pH5 to pH3.5, in the absence of compound No. 2 ( Figure 4) and in the presence of compound No. 2 ( Figure 5).
- Example 24 Conservation of the enzymatic activity of ⁇ -galactosidase.
- the solubilizing agents of the invention do not have a denaturing effect on ⁇ -galactosidase since the latter retains its enzymatic activity when it is in the presence of the solubilizing agents of the invention .
- a commercial suspension (760 units per ml) of ⁇ -galactosidase is used which is diluted 250 times in a buffer: HEPES-NaOH (pH 7.8) 50mmol / l, NaCl 200mmol / l, DTT lmmol / 1, Mg Cl 2 lmmol / 1 containing lmol / 1 of compound No.
- Example 1 of Example 1 as a solubilizing agent.
- the solution is incubated for 15 h at 8 ° C., it is then diluted 25 times, then its enzymatic activity is measured, at 37 ° C., by detection of the hydrolysis of o-nitrophenyl ⁇ -D-galactopyranoside (ONPG ) (2mmol / l), which results in an increase in optical density to 405nm.
- ONPG o-nitrophenyl ⁇ -D-galactopyranoside
- control 1 a buffer not containing the solubilizing agent of the invention
- control 2 determining the enzymatic activity of a commercial suspension of diluted ⁇ -galactosidase directly without incubation
- Example 28 Effects of the concentrations of compound n ° 2 on the activity of ⁇ -galactosidase.
- FIG. 7 illustrates the activity of ⁇ -galactosidase (in ⁇ mol of hydrolysis substrate (ONPG) per mg of enzyme) as a function of the concentration of compound No. 2 (in mol / 1) of the medium for determining the enzymatic activity.
- a commercial suspension of alkaline phosphatase is diluted 250 times in a tris-HCl buffer (pH 8.8) 50mmol / l, NaCl 200mmol / l, ZnCl 2 0, lmmol / l, MgCl 2 0.5mmol / l containing 0, 0.2 or lmol / 1 of compound No. 2.
- These solutions are incubated for 15 h at 8 ° C., then they are diluted and their activity is measured at 37 ° C., in the presence of increasing concentrations of compound No. 2, by detection of the hydrolysis of p-nitrophenyl phosphate (PNP) at 1.3 mmol / l, which results in an increase in the optical density at 405 nm.
- PNP p-nitrophenyl phosphate
- Example 30 Renaturation of ⁇ -galatosidase.
- a commercial suspension of ⁇ -galactosidase is diluted 250 times in a solution containing 8mol / l of urea, 50mmol / l of dithiothreitol, lmmol / 1 of MgCl 2 and 50mmol / l of tris-hydroxymethylam omethane-HCl (pH 7.8 ).
- the solution is left for 30 mm at 20 ° C., then it is dialyzed for 18 h against a lmmol / 1 solution of MgCl 2 and 50mmol / l of tris-hydroxymethylaminomethane-HCl (pH 7.8) in the presence of lmol / 1 of compound No. 2 or in the absence of this compound.
- the enzymatic activity is then determined as in Example 24.
- the starting buffer contains 20mmol / l of HEPES-NaOH (pH 7.4), lmmol / 1 of ethylene diamine tetracetate.
- the limit buffer comprises 20mmol / l of HEPES-NaOH (pH 7.4), lmmol / 1 of ethylene diamine tetracetate and lmol / 1 of NaCl.
- the buffer flow rate is lml / min.
- the column is balanced with 10 ml of buffer A, then 0.5 ml of a sample produced by digestion of fibronectin (0.5 mg / ml) with thermolysin is loaded as described by Sekiguchi et al in Journal of Biological Chemistry, vol 260, n ° 8, 1985, p. 5105- 5114. Elution is carried out either by a complex gradient from 0 to 100% of buffer B in A in 20 mm, or by a complex gradient from 0 to 100% of buffer B in buffer A in 20 min, the two buffers containing plus each lmol / 1 of compound 2.
- fractions from the column are checked by analysis by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate.
- Figure 9 illustrates the results obtained during this analysis.
- the left part of this figure refers to the fractions obtained in the absence of compound No. 2, while the right part refers to the fractions obtained in the presence of compound No. 2.
- HEPES hydroxyethyl piperazine ethane sulfonic acid
- DTT dithiothreitol
- PMSF phenylmethyl sulfonyl fluoride
- SDS sodium dodecyl sulfate
- HEPES hydroxyethyl piperazine ethane sulfonic acid
- DTT dithiothreitol
- EDTA ethylene diamine tetracetic acid
- PMSF phenylmethyl sulfonyl fluoride
- CHAPS 3 (3cholamidopropyl) dimethyl ammonio propane sulfonate
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE0739334T DE739334T1 (de) | 1994-01-12 | 1995-01-11 | Solubilisierungsmittel für biomoleküle, bestehend aus nicht-tensidischen zwitterionen und ihre verwendung |
JP7518875A JPH09511735A (ja) | 1994-01-12 | 1995-01-11 | 非洗浄性双性イオンからなる生物分子溶解剤およびその使用 |
EP95907027A EP0739334A1 (fr) | 1994-01-12 | 1995-01-11 | Agents de solubilisation de biomolecules constitues par des zwitterions non detergents et leurs utilisations |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9400241A FR2714906B1 (fr) | 1994-01-12 | 1994-01-12 | Agents de solubilisation de biomolécules constitués par des zwitterions non détergents et leurs utilisations. |
FR94/00241 | 1994-01-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995019339A1 true WO1995019339A1 (fr) | 1995-07-20 |
Family
ID=9458941
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR1995/000035 WO1995019339A1 (fr) | 1994-01-12 | 1995-01-11 | Agents de solubilisation de biomolecules constitues par des zwitterions non detergents et leurs utilisations |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0739334A1 (fr) |
JP (1) | JPH09511735A (fr) |
DE (1) | DE739334T1 (fr) |
ES (1) | ES2099052T1 (fr) |
FR (1) | FR2714906B1 (fr) |
WO (1) | WO1995019339A1 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1021709A1 (fr) * | 1996-12-05 | 2000-07-26 | Macquarie Research Ltd. | Procedes de separation par electrophorese |
US6593107B1 (en) | 1997-10-08 | 2003-07-15 | Genetics Institute Llc | Methods of refolding proteins |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4372888A (en) * | 1980-08-26 | 1983-02-08 | The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services | Nondenaturing zwitterionic detergents |
EP0141879A1 (fr) * | 1983-10-18 | 1985-05-22 | Victor A. Bernstam | Compositions surfactantes et méthodes pour la clarification et le partage de spécimens aqueux contenant des lipides |
EP0471293A2 (fr) * | 1990-08-15 | 1992-02-19 | Abbott Laboratories | Réactif de solubilisation pour des spécimens biologiques |
EP0494686A1 (fr) * | 1991-01-11 | 1992-07-15 | Millipore Corporation | Procédé pour l'exécution de séparations de haute efficacité par électrophorèse capillaire |
-
1994
- 1994-01-12 FR FR9400241A patent/FR2714906B1/fr not_active Expired - Fee Related
-
1995
- 1995-01-11 WO PCT/FR1995/000035 patent/WO1995019339A1/fr not_active Application Discontinuation
- 1995-01-11 EP EP95907027A patent/EP0739334A1/fr not_active Withdrawn
- 1995-01-11 ES ES95907027T patent/ES2099052T1/es active Pending
- 1995-01-11 DE DE0739334T patent/DE739334T1/de active Pending
- 1995-01-11 JP JP7518875A patent/JPH09511735A/ja active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4372888A (en) * | 1980-08-26 | 1983-02-08 | The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services | Nondenaturing zwitterionic detergents |
EP0141879A1 (fr) * | 1983-10-18 | 1985-05-22 | Victor A. Bernstam | Compositions surfactantes et méthodes pour la clarification et le partage de spécimens aqueux contenant des lipides |
EP0471293A2 (fr) * | 1990-08-15 | 1992-02-19 | Abbott Laboratories | Réactif de solubilisation pour des spécimens biologiques |
EP0494686A1 (fr) * | 1991-01-11 | 1992-07-15 | Millipore Corporation | Procédé pour l'exécution de séparations de haute efficacité par électrophorèse capillaire |
Non-Patent Citations (1)
Title |
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P.C. MALONEY, ET AL.:: "Functional reconstruction of prokaryote and eukaryote membrane proteins", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS,, vol. 269, no. 1, 15 February 1989 (1989-02-15), pages 1 - 10 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1021709A1 (fr) * | 1996-12-05 | 2000-07-26 | Macquarie Research Ltd. | Procedes de separation par electrophorese |
EP1021709A4 (fr) * | 1996-12-05 | 2006-01-25 | Macquarie Res Ltd | Procedes de separation par electrophorese |
US6593107B1 (en) | 1997-10-08 | 2003-07-15 | Genetics Institute Llc | Methods of refolding proteins |
US6593106B1 (en) | 1997-10-08 | 2003-07-15 | Genetics Institute Llc | Methods of refolding proteins |
US6596511B1 (en) | 1997-10-08 | 2003-07-22 | Genetics Institute Llc | Methods of refolding proteins |
US6632635B1 (en) | 1997-10-08 | 2003-10-14 | Genetics Institute | Methods of refolding proteins |
Also Published As
Publication number | Publication date |
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FR2714906B1 (fr) | 1996-02-02 |
ES2099052T1 (es) | 1997-05-16 |
JPH09511735A (ja) | 1997-11-25 |
DE739334T1 (de) | 1997-06-05 |
EP0739334A1 (fr) | 1996-10-30 |
FR2714906A1 (fr) | 1995-07-13 |
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