US3915839A - Apparatus for isoelectric focusing - Google Patents

Apparatus for isoelectric focusing Download PDF

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US3915839A
US3915839A US356307A US35630773A US3915839A US 3915839 A US3915839 A US 3915839A US 356307 A US356307 A US 356307A US 35630773 A US35630773 A US 35630773A US 3915839 A US3915839 A US 3915839A
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column
focusing
invention defined
electrode
walls
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Svante Harry Rilbe
Jarl Sune Pettersson
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Pfizer Health AB
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LKB Produkter AB
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing

Definitions

  • the present invention relates to a method in separation of components of a sample solution by isoelectric focusing in a focusing chamber which is subject to a direct current electrical field, and to an apparatus for accomplishing the method.
  • isoelectric focusing is an electrophoretic method of separation, based on differences in isoelectric points of the sample components to be separated, and which is performed in an electrical current path having a varying pH.
  • Isoelectric focusing is generally characterized in that after equilibrium has been achieved, the separated substances are located in that point in the pH- gradient which corresponds to the isoelectric point of each substance, respectively, and that each substance there has zero mobility.
  • a survey of isoelectric focusing as separation method has been given by Haglund in Methods of Biochemical Analysis, 19, page 1-104, John Wiley & Sons (1971).
  • the pH-gradient requisite in isoelectric focusing, can be obtained in two principally different ways.
  • the pH-gradient could be artificial or natural.
  • the artificial pH-gradient is achieved by a mechanically obtained mixture in gradually changed proportion of solutions of varying pH.
  • the natural pH-gradient is obtained by the influence of the electrical field on a number of ampholytical substances having different isoelectric points.
  • the natural pH-gradient has the advantage after having been formed by influence of the electrical field to be stabilized thereby.
  • the theory of natural pH-gradients was discussed by Svensson in Acta Chem. Scand. 15, page 325-341 (1961), and in Acta Chem. Scand. 16, page 456-466 (1962).
  • carrier ampholytes In order to achieve practically a natural pH-gradient, suitable so called carrier ampholytes are required, the isoelectric points of which are narrowly and evenly distributed over the desired pH-interval and which possess in their respective isoelectric points buffering as well as electrically conducting properties.
  • the lack of good carrier ampholyte was the limitation of this method until such substances were made available by an invention, made by Vesterberg, disclosed in the U.S. Pat. No. 3,485,736 (corresponding to Swedish Pat. No. 314,227).
  • the disclosed mixture of carrier ampholytes are marketed by LKB-Sher AB, Bromma, under the Trade Mark Ampholine.
  • the present invention can be performed by utilization of an artificial or a natural pH-gradient. It is preferred by the performance of the present invention to use a natural pH-gradient.
  • the isoelectric focusing is carried out in a density gradient.
  • This method implies that the relatively small density differences depending on temperature differences as well as those depending on the concentrations of the zone components are drowned in considerable greater, artificial density differences.
  • the density gradient should be parallel or anti-parallel to the applied electrical field.
  • the density of the solution is continuously increasing downwards. This is achieved by using a heavier and a lighter solution, the proportion of the heavier solution increasing downwards in the column in which the isoelectric preparation is performed, while the proportion of the lighter solution decreases. Both solutions contain electrolytes, the heavier one in addition sugar to an amount of 500 grams/liter. The total density difference will amount to about 0.2 grams/cm.
  • Substances which are suitable for separation by isoelectric focusing are ampholytes.
  • An important group of such substances are proteins.
  • a criteria of a good separation is the resolving power of the separation method.
  • resolving power is directed towards the smallest difference in isoelectric points of two components of the sample solution, which will result in two perceivable zones after focusing.
  • dpH/dx should consequently be given as low a value as possible.
  • D and du/dpH are constant for a given substance, while an increase in E theoretically should result in an increased resolution.
  • An increase in E will entail an increased heat evolvation in the sample solution which would imply increased risk of heat convection.
  • E is limited by the ability of the prevailing density gradient to cover the convection caused by heat evolvation.
  • the pH-slope is made very flat, this will entail that also the density change is distributed over a longer distance, which will decrease its convection stabilizing capacity. Consequently, the electrical field strength has to be kept sufficiently low in order that the generation of heat convection is avoided. This will result in low ion migration velocities, which will bring about long separation time.
  • the pH-gradient extended over a long distance moreover will entail long migration paths for the majority of the samples components, which will contribute to a prolonged focusing time. Consequently, the best possible resolution is achieved at the cost of slow focusing.
  • the purpose of the invention is accomplished by performing a focusing step with a mean cross-section of the focusing chamber, perpendicular to the electrical direct current field which is smaller than the corresponding mean cross-section in the nearest preceding focusing step.
  • More closely one focusing step is performed with a greater relation between column height, i.e. that distance in the column over which the electrical field is acting, and cube root of the column volume that in the nearest preceding focusing step.
  • the simplest form of a column device having varying mean cross-section perpendicular to the electrical field consists of one parallelepipedical column with the linear measures a, b and c, a b c, in which the isoelectric separation according to the invention can be accomplished in three steps, the first one with c, the second one with b and the third one with a as vertical dimension.
  • the intermediate step can be omitted.
  • Such a column requires different electrodes for different spatial orientations. Therefore, the electrodes have to be detachable.
  • the invention can also be performed by using a column with vertical walls made by an elastic material, like rubber, which permits a continuous varying of the mean cross-section perpendicular to the electrical field with retained column volume.
  • the invention can finally be performed by using at least two columns having different mean cross-section perpendicular to the electrical field, situated in sequence, one above another, and provided with connecting tubes between the top of a lower situated column and the bottom of the next higher situated one.
  • the connecting tubes should be mainly vertical in order to allow, after completed prefocusing in one column, a transport free of convection of the column content to the next column for fine focusing.
  • the outflow can be improved by making top and bottom conical, with the outlet tube situated in the apex of the cone. This is, however, no good settlement, as it will entail a higher field strength and current density in the peripheral parts of the column than in its central parts. This will lead to a horizontal temperature gradient followed by a horizontal density gradient contribution which will easily result in thermical convection.
  • the tilting of the column 90 before emptying should be carried out by means of suitable mechanical means, preferably by motor operation, and the tilting should not be carried out faster than during a few minutes. Manual turning could hardly be accomplished sufficiently slowly and carefully.
  • the fluid transport from a prefocusing column to a fine focusing column of the same volume as the prefocusing column could be carried out upwards as well as downwards.
  • a mainly vertical connection tube between the uppermost point of the prefocusing column and the bottom of the fine focusing column is utilized.
  • the upward transport is accomplished by pumping into the bottom of the prefocusing column a fluid with at least as great a density as the greatest density in the sample solution. The column content is thereby lifted with a minimum of disturbance into the originally empty fine focusing column.
  • connection tube of the prefocusing column it is possible without use of a fluid pump to allow the lower connection tube of the prefocusing column to dip into a supply of a fluid with at least as great a density as the greatest density in the sample solution, whereupon a suitable vacuum is applied to the fine focusing column. The column content will then be sucked up from the prefocusing column into the fine focusing column.
  • the transportation velocity is adjusted to a low value, either by adjusting the vacuum in the upper column or by throttling of one of the connection tubes.
  • the fine focusing column is first filled with a fluid with at least as great a density as the greatest density in the sample solution. Then a mainly vertical tube connection, free of air, is established between the upper end of the fine focusing column and the bottom of the prefocusing column. By opening of a bottom valve of the fine focusing column the fluid content of the prefocusing column is caused to flow down spontaneously into the fine focusing column with a minimum of disturbance.
  • the electrode system could be designed in many different ways. To place the electrodes in separate electrode vessels of considerable volume compared to the column volume was introduced into the classical electrophoresis research by Michaelis in Biochem. Z., 16, page 81 (1909) and meant at that time an important progress as possible disturbing influence of electrode reaction products in the column thereby was prevented. Such disturbing influence could be brought about by ion migration, by diffusion or by convection from the electrodes to the column.
  • the principle of electrodes in separate electrode vessels could advantageously be utilized also in the present invention. In that case the electrode vessels should be in electrolytically conducting connection to the column device, one to its top and the other to its bottom. In order to prevent undesired ion migration throughout the column the anolyte must consist of an acid or of a mixture of acids, while the catholyte must consist of a base or of a mixture of bases.
  • the column content is delimited from anolyte and catholyte by convection breaking membranes, permeable to electrical current.
  • convection breaking membranes permeable to electrical current.
  • Such membranes could consist of glass filter discs, other porous discs, cellophane, relatively thick paper membranes, animal membranes, natural or synthetic gel discs, etc. These membranes should be strictly horizontal and should completely cover the cross-section of the column without leakage.
  • connection tubes between electrode vessels and column could therefore be rather slender and do not require particular cooling.
  • an electrode In order that an electrode should be non-gasing it is required that at the anode there is a reducing agent sufficiently strong to prevent evolvation of oxygen and at the cathode an oxidation agent sufficiently strong to prevent evolvation of hydrogen gas.
  • a reducing agent sufficiently strong to prevent evolvation of oxygen
  • an oxidation agent sufficiently strong to prevent evolvation of hydrogen gas.
  • One exception from this rule is constituted by palladium which in pure form or as a silver alloy is able to absorb large quantities of hydrogen.
  • a palladium electrode, saturated with hydrogen could be utilized as a non-gasing anode as hydrogen in that case is present in the metal as a reducing agent. This property of palladium is well known since long, but it was not made use of in electrophoretical apparatus until 1960 by Neihof and Schuldiner (Nature 185, page 526 (1960)).
  • the hydrogen absorbing ability of palladium is also utilized.
  • the reducing agent which has to be present at a nongasing anode, could either be the metal itself or the anolyte acid or some non-electrolyte which also is part of the anolyte.
  • An example of the metal itself being reducing agent is a silver electrode in an anolyte of hydrogen halide acid, yet not hydrofluoric acid.
  • the silver halide, formed at the anode oxidation, is almost completely unsoluble and will consequently not give rise to any migration of silver ions into the column.
  • Another example is a lead anode in an anolyte environment of sulphuric acid.
  • a third example is mercury in hydrogen halide acid environment, but mercury could only be used as a bottom electrode.
  • anolyte acid as reducing agent is hydrogen bromide acid or hydrogen iodide acid in contact with a noble metal or carbon anode.
  • a noble metal or carbon anode used ammonium bromide in contact with a carbon anode.
  • the oxidation at the anode generates free liquid bromide, which however may diffuse into the column and cause damage on proteins there.
  • a more harmless, reducing acid is ascorbic acid.
  • the oxidating agent requisite at a non-gasing cathode, can also be present on the cathode itself or in the catholyte.
  • Silver covered by an adhering layer of silver halide, is a known example, but such a cathode, when working, will evolve halide ions which will migrate into the column.
  • a bottom electrode of mercury covered by a layer of calomel works in a corresponding way.
  • Oxidating, soluble bases are not known. Consequently, if the oxidation agent should be present in the catholyte it has to be a non-electrolyte. Only palladium is able to function as non-gasing cathode without generating foreign anions which will migrate into the column.
  • FIGS. 1 and 1a are, respectively, longitudinal and transverse cross-sectional elevations of a preferred form of one form of separation chamber
  • FIG. 2 is a vertical cross-section of a modified form of prefocusing column combined with a final focusing column;
  • FIG. 3 is a cross-section of a column similar to FIG. 2 but having a modified cooling system
  • FIG. 4 is similar to FIGS. 2 and 3, but modified to place the final column below the prefocusing column.
  • FIG. 1 shows a parallelepipedical combination column 14 comprising a focusing chamber 15 having internal measures a, b, and 0 length units, a b c.
  • a focusing chamber 15 having internal measures a, b, and 0 length units, a b c.
  • Each of the two smallest walls, internally bc units of surface, is provided with a threaded opening 5,6 in which alternatively a threaded stopper, a threaded tube or a threaded electrode attachment could be inserted.
  • the attachment of detachable electrodes constitutes the most difficult problem. Therefore, the construction shown in the figure is restricted to the realization of current transport along the dimensions 12 and a, which then should be vertical dimensions as the current direction has to be parallel or antiparallel to the density gradient.
  • the column stands on a face having the internal measures ac units of surface, and in that position the prefocusing is accomplished.
  • the column is situated on a face having the internal measures be units of surface, and in this position the final focusing is accomplished.
  • the column could also be layed on a face having the internal measures ab units of surface, i.e. with c as vertical dimension.
  • This position is suitably utilized at preparation of the density gradient as described by the Inventors in Separation Science 3, page 535 (1968).
  • a small number of solutions having rising density are layered one under the other in a column, whereupon the column is turned down to a horizontal position in order to enlarge the diffusion area as well as the concentration gradient.
  • the mass transport due to free diffusion thereby is increased to such an extent that only a short while is required for development of a continuous density variation in the column.
  • the problem of the bottom electrode could be solved very easily by using mercury, which always is spreading over the bottom independent of the spatial orientation of the column.
  • Mercury 7 as an anode is non-gasing if it is present in an environment of hydrogen halide acid or sulphuric acid, as difficultly soluble mercury I salts are formed.
  • Mercury as cathode is non-gasing in an environment of alkali hydroxides due to the formation of alkali metal amalgam.
  • the external connection of a mercury electrode is also most simple.
  • a platinum wire 8 will suffice, going through the column wall and arriving at an arbitrary corner of the column.
  • the lead-through is intended to be bent at an angle and leading through a column face of internal measures ab units of surface to make possible that the column is placed upon a column face of internal measures ac units of surface.
  • Every non-gasing electrode must have a large area in order to keep the current density as low as possible.
  • a non-gasing top electrode consequently must have an area of the size of ac units of surface.
  • the most suitable material is palladium-silver-alloy as cathode and the same alloy, saturated with hydrogen, as anode. Any fluid space between such an electrode and the column wall is non-desirable. Then it remains nothing but to coat this column wall with palladium-silver-alloy, and then the whole wall has to be detachable. A constructively simple way to make this is shown in the figure.
  • the column wall 9 itself, made of plastic, having a T- shaped cross-section, is fastened by means of a number of screws 10 and is tightened by means of a O-ring 11.
  • the end wall has a cut out slit 13 having the electrode 12 protruding out of it to make possible to be connected to the voltage source. After completed prefocusing, the current is disconnected and all the screws 10 are loosened. The electrode 12 then can be pulled out whereupon all the screws 10 are once again tightened.
  • the column then is carefully turned upright in order that a will become vertical dimension.
  • a platinum wire is introduced, fastened in a threaded electrode attachment provided with a gas outlet hole, whereupon voltage could be applied for final focusing.
  • the top electrode is replaced by an attachment having a tight threaded stopper, and the stopper which till then has been situated in the treaded hole 5 is replaced by a threaded outlet tube having a regulating valve.
  • the stopper in the hole 6 now can be removed.
  • the column is slightly tilted in order that the mouth of the hole 5 will be the lowest point of the column. After that the column can be emptied from its content, which is at that divided into fractions for pH-measurement and chemical and/or biological analysis.
  • the column can alternatively be made of quartz, whereby UV-absorption analysis can be accomplished directly in the column during the performing of focusing, which is of great advantage.
  • the fastening of the column wall 9 by screws then must be substituted by a device for clamp fastening, but besides, the same construction can be retained.
  • Two different sensitivity degrees in the optical analysis can be achieved by allowing the light to pass the path b or the path 0 in the column. The shift between these degrees of sensitivity is simply accomplished by turning the column
  • To utilize mercury as anode can be associated with certain risks if it is considered that mercury ions could go into solution and migrate through the column and transform the proteins in the column by complex formation.
  • mercury I halides are as sparingly soluble that they would hardly give rise to deleterious concentrations of mercury I ions, but at the anodical oxidation of mercury also small amounts of mercury II salts, which are soluble are formed. However, these are so weakly dissociated that their cathodical migration will be very slow. Anyhow, there are doubtless cases in which mercury anode is completely inconceivable.
  • the combination column then must be used with the cathode in the bottom and a sufficient reserve of alkali metal ions, or mercury as an electrode has to be completely abandoned. In the latter case the column construction has to be modified.
  • the palladiumsilver-anode then should be saturated with hydrogen and the palladium-silver-cathode be free of hydrogen, and both the electrodes should be removed after prefocusing.
  • a remaining problem then is how to realize a non-gasing bottom electrode with an area of be units of surface during the final focusing.
  • the best way to achieve this is to prepare an extra column wall having the internal measure ac units of surface and in one end of this constructional element attach a pal]adium-silver-electrode, which is slightly smaller than be units of surface.
  • FIG. 2 shows a column device comprising a prefocusing column 21 and a final focusing column 25in the position of the device during the transport of prefocused material from one column to the other and during the final focusing.
  • the device is turned 90 around an axis perpendicular to the plane of the paper so that the final focusing column 25 then will have a horizontal axis.
  • the final focusing column 25 in this example is represented by the above mentioned apparatus, marketed by LKB-Helper AB. As described above, this apparatus comprises in addition to the column noble metal electrodes, a central gas outflow tube as well as a cooling jacket.
  • prefocusing column The central part of prefocusing column is the focusing chamber 21 itself, limited at the top and at the bottom by two electrolytically conducting but convection breaking membranes 24, e.g. glass filter discs. Laterally the focusing chamber is restricted by vertical walls, e.g. by a short elliptical or circular cylinder. In two diametrically opposite points there are threaded holes 30, 31 in which alternatively threaded stoppers or tubes can be inserted. During prefocusing there is in the hole 30 a stopper, in the hole 31 a tube which via a cock 32 forms the connection to the final focusing column.
  • anolyte chamber 22 or 23 Outside the membrane 24 there is an anolyte chamber 22 or 23 and a catholyte chamber 23 or 22 which via tubes and circulation pumps 29 is connected each to one electrode vessel 26,28 containing each an electrode 27.
  • Each tube-pipe comprises a coil, immersed into a cooling reservoir, which details, however, are not shown in the figure.
  • a suitable clamping device which is neither shown, is holding in a leakage free manner anolyte chamber, focusing chamber, and catholyte chamber together.
  • this column device By operation of this column device first the membranes 24 are thoroughly moistened in order to be im' permeable to air. Then into the hole 30 a tube is inserted which is connected to a density gradient mixing device, and the cock 32 is opened. With the whole device in final focusing position a vacuum is applied to the final focusing column in order that the prefocusing chamber 21 is filled with a solution of desired density variation. The cock 32 is closed, and the tube in the hole 30 is substituted by a stopper. Whole the device is turned around an axis perpendicular to the plane of the paper, the electrode vessels are filled with anolyte and catholyte, respectively, and the circulation pumps 29 are started. Voltage is applied to the electrodes for prefocusing.
  • the device After completed prefocusing the current is disconnected, and the device is carefully turned 90 around an axis perpendicular to the plane of the paper. Anolyte and catholyte then are allowed to flow out through the gas outlet holes of the electrode attachments. It is also possible to completely remove the electrodes with their attachments.
  • the stopper in the hole 30 is substituted by a tube, filled with and dipping into a fluid having greater a density than the solution in the bottom of the column.
  • This tube should also be provided with a cock or, if rubber or plastic tubes are used, a ligature clamp. With the latter one closed, the cock 32 is opened.
  • the column content in the chamber 21 is replaced by the said heavier fluid, and the prefocused material then will ascend into the final focusing column, the cooling jacket of which during this operation being throughflown by cooling medium.
  • voltage is applied to the final focusing column after the cock 32 has been closed. The prefocusing column is removed, and after the final focusing the column content can be fractioned by careful outflow through the cock 32.
  • FIG. 3 shows a column device distinguished from that one shown in FIG. 2 only concerning the cooling system.
  • the anolyte and catholyte chambers are made larger in order that there is room for cooling coils 33,34 through which cooling medium from a cooling reservoir is made to circulate.
  • This device has the advantage compared to that one shown in FIG. 2 that the circulation pumps are not under a voltage.
  • the electrodes 35 can be attached in rather slender tubes 36,37 which are centrally leading out into the anolyte and catholyte chambers.
  • the utilization of column devices according to FIG. 2 and FIG. 3 have in common that the anolyte should consist of a solution of a strong acid and the catholyte of a solution of a strong base. Their concentrations should be between 0.1 and l equivalents per liter. Their conductivities then will be several l0-powers greater than that one prevailing in the central parts of the column at focusing. This is why it is possible in FIG. 2 and 3 to have as narrow tubes for supplying of current to the anolyte and catholyte chambers. There is no risk of uneven current density caused by the local falling out of the tubes into the anolyte and catholyte chambers.
  • FIG. 4 shows how a column device could be arranged in which the electrodes are in direct contact with the prefocusing column and in which the fluid transport between the two columns is carried out from the top downwards.
  • the figure is showing the device being in its position during fluid transport from one column to the other and during final focusing.
  • the device is turned 90 around an axis perpendicular to the plane of the paper, in order that the final focusing column 48 then will have a horizontal axis.
  • the final focusing column is represented by the above mentioned apparatus marketed by LKB-Sher AB.
  • this apparatus comprises, besides the column itself, noble metal electrodes, a centrally situated gas outflow tube, and a cooling jacket.
  • these constructional details are known as such and are not making part of the invention, only the outer countours of this column have been drawn in the figure. The volumes of the two columns are equal.
  • the prefocusing column 52 is at its top and bottom limited by two non-gasing electrodes 42 and 46, each joint by soldering to one external wall of two cooling chambers 47 and 49, each provided with two pipe sockets 41. Laterally the prefocusing column is restricted by vertical walls 44, e.g. being a short elliptical or circular cylinder. In two diametrically opposite points there are threaded holes 45,50 in which alternatively threaded stoppers or tubes could be inserted. During prefocusing there is in the hole 45 a stopper, in the hole 50 a tube which via a cock 51 forms the connection to the final focusing column 48.
  • the prefocusing column is tightened by means of O-rings 43 combined with a clamping device acting on the outer walls of the two cooling chambers.
  • the final focusing column When operating this column device it is first placed in final focusing position (as in the figure), the final focusing column is filled with a fluid with greater a density than the bottom solution to be used in the prefocusing column, whereupon the bottom cock of the final focusing column and the cock between the columns are closed.
  • the prefocusing column then is filled with an amfolyte solution containing the protein sample, out of a density gradient mixer, whereupon the hole 45 is closed by means of a stopper.
  • the column device then is turned slowly 90 around an axis perpendicular to the plane of the paper, the cooling water flow is started and electrical voltage is applied for prefocusing. After completed prefocusing the current is disconnected and the column device is turned backwards 90.
  • the cocks in both ends of the final focusing column are opened, and the stopper in the hole 45 is removed.
  • the flow rate downwards is adjusted to a suitable, low value.
  • Apparatus for the separation of components in a sample solution by isoelectric focusing in which said focusing is performed in at least two successive focusing steps comprising, closed container means to be disposed in a first position during said first focusing step and in a second position during the second focusing step, the interior thereof defining a focusing chamber, at least two pairs of oppositely disposed walls, the mean area of one pair of said walls being less than the mean area of the other pair of said walls, the mean distance between said one pair of walls being greater than the mean distance between said other pair of walls, an electrode system for selectively generating a vertical direct current electrical field across said sample between the respective inner surfaces of each pair of said two pairs of oppositely disposed walls in either of said first or second positions of the container means.
  • Apparatus according to claim 1 which includes means for externally cooling said focusing chamber.
  • said electrode system includes a separate vessel contianing an electrode and an electrolyte solution therein, said separate vessel and said closed container means being separated from each other by a convection breaking electrolytically conducting membrane, said membrane defining at least one of said two pairs of walls, said membrane separating the sample solution from the electrolytic solution.
  • said means for cooling said electrolytic solution includes conduit means in said separate vessel for conducting a cooling medium in heat exchanging relationship through said electrolytic solution.
  • said electrode system includes at least one electrode element in direct contact with said sample solution in one position of said container means.
  • said electrode element comprises palladium
  • said mercury electrode element is disposed at the bottom of said focusing chamber on a respective one of said two pairs of oppositely disposed walls in either of said first and second positions of the container means, said means also including means for cooling said mercury.
  • At least one wall of at least one of said two pairs of walls includes a flat electrode extending over the entire area of the inner surface of said one wall.
  • said closed container means includes a removable section, the inner surface of said section defining said one wall, and means for detachably connecting said section in fluid tight engagement with the remainder of the container means.

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4533447A (en) * 1983-06-13 1985-08-06 Meldon Jerry H Apparatus for and method of isoelectric focussing
US4670119A (en) * 1985-10-25 1987-06-02 Hurd Stanley M Isoelectric focusing device and process
US4963236A (en) * 1989-03-08 1990-10-16 Ampholife Technologies Apparatus and methods for isoelectric focusing
US5160594A (en) * 1989-03-08 1992-11-03 Board Of Regents Of The University Of Texas System Apparatus and methods for isoelectric focusing of amphoteric substances incorporating ion selective membranes in electrode chambers
US5599506A (en) * 1992-05-28 1997-02-04 Aligena Ag Immobilized buffered gels and membranes of hydroxy groups containing polymers
US6537434B1 (en) 2000-07-21 2003-03-25 Large Scale Proteomics Corporation First dimension electrophoresis separation method and apparatus
US20040163956A1 (en) * 2003-02-26 2004-08-26 Milan Bier Variable-volume disposable isoelectric focusing cell and method of isoelectric focusing
US20080019955A1 (en) * 2003-12-30 2008-01-24 Bioactive Polymers Ab Surface Protection of Exposed Biological Tissues
US9375518B2 (en) 2003-12-30 2016-06-28 Bioactive Polymers Ab Surface protection of exposed biological tissues

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US4533447A (en) * 1983-06-13 1985-08-06 Meldon Jerry H Apparatus for and method of isoelectric focussing
US4670119A (en) * 1985-10-25 1987-06-02 Hurd Stanley M Isoelectric focusing device and process
US4963236A (en) * 1989-03-08 1990-10-16 Ampholife Technologies Apparatus and methods for isoelectric focusing
US5160594A (en) * 1989-03-08 1992-11-03 Board Of Regents Of The University Of Texas System Apparatus and methods for isoelectric focusing of amphoteric substances incorporating ion selective membranes in electrode chambers
US5599506A (en) * 1992-05-28 1997-02-04 Aligena Ag Immobilized buffered gels and membranes of hydroxy groups containing polymers
US6537434B1 (en) 2000-07-21 2003-03-25 Large Scale Proteomics Corporation First dimension electrophoresis separation method and apparatus
US20040163956A1 (en) * 2003-02-26 2004-08-26 Milan Bier Variable-volume disposable isoelectric focusing cell and method of isoelectric focusing
WO2004077039A1 (en) * 2003-02-26 2004-09-10 Milan Bier Variable-volume disposable isoelectric focusing cell
US6793791B2 (en) * 2003-02-26 2004-09-21 Milan Bier Variable-volume disposable isoelectric focusing cell and method of isoelectric focusing
US20080019955A1 (en) * 2003-12-30 2008-01-24 Bioactive Polymers Ab Surface Protection of Exposed Biological Tissues
US8461104B2 (en) 2003-12-30 2013-06-11 Bioactive Polymers Ab Surface protection of exposed biological tissues
US9375518B2 (en) 2003-12-30 2016-06-28 Bioactive Polymers Ab Surface protection of exposed biological tissues

Also Published As

Publication number Publication date
DE2324521A1 (de) 1973-11-29
FR2184858A1 (enrdf_load_stackoverflow) 1973-12-28
DE2324521B2 (de) 1978-09-07
GB1437377A (en) 1976-05-26
DE2324521C3 (enrdf_load_stackoverflow) 1979-08-02
SE361829B (enrdf_load_stackoverflow) 1973-11-19
FR2184858B1 (enrdf_load_stackoverflow) 1977-02-18

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