WO2004072236A2 - Procedes et compositions pour electrophorese sur gel de cube de sodium dodecyl sulfate-polyacrylamide 3-d - Google Patents

Procedes et compositions pour electrophorese sur gel de cube de sodium dodecyl sulfate-polyacrylamide 3-d Download PDF

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WO2004072236A2
WO2004072236A2 PCT/US2004/002953 US2004002953W WO2004072236A2 WO 2004072236 A2 WO2004072236 A2 WO 2004072236A2 US 2004002953 W US2004002953 W US 2004002953W WO 2004072236 A2 WO2004072236 A2 WO 2004072236A2
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dimension
separation
sds
gel
proteins
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PCT/US2004/002953
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WO2004072236A3 (fr
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Bao-Shiang Lee
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The Board Of Trustees Of The University Of Illinois
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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
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • the invention relates to the field of electrophoretic separations of macromolecules and in particular, to a technique for the electrophoretic separation used in the analysis of proteins.
  • Electrophoresis is one of the most widely used separation techniques in the biologically related sciences. This technique separates molecular species such as peptides, proteins, and oligonucleotides (analytes) by causing them to migrate at different rates in a separation medium under the influence of an electric field.
  • the separation medium can be a buffer solution, or a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide.
  • gel separation medium separation of analytes is partly based on their molecular sizes as the analytes are sieved by the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size which depends in part on the concentration of the polymer in the gel.
  • IEF is almost exclusively the first separation dimension, h IEF, amphoteric molecules such as proteins are separated by electrophoresis in a pH gradient generated between a cathode and an anode.
  • IEF takes advantage of the fact that each protein has a characteristic pH at which it is electrically neutral. This characteristic pH is the isoelectric point (pi) of the protein.
  • pH is the isoelectric point (pi) of the protein.
  • electrophoresis medium a solution or a gel. If a sample component has a net negative charge, it migrates towards the anode. During migration, the negatively charged sample encounters a progressively lower pH, thus becoming more positively charged. Eventually, the pi is reached where the net charge of the sample component is zero.
  • Carrier ampholites are polyamino- polycarboxic acids having gradually differing pi values. Ampholite mixtures are available in various nanow and broad pH ranges. Typically, an anti-convective media such as polyacrylamide or agarose is used. It is also possible to immobilize pH gradients on a suitable matrix such as polyacrylamide or ampholite strips. With immobilized pH gradients, LEF routinely provides a resolution of 0.1 to 0.01 pi units.
  • capillary dimensions i.e. dimensions less than 0.2 mm ID.
  • IEF separations can be carried out in free solution or in entangled polymer networks.
  • SDS-PAGE the second separation dimension in 2-D SDS PAGE is typically carried out by SDS-PAGE.
  • SDS-PAGE involves complex relationships among several factors. These factors include separation length, gel composition, gel pore size, electric field strength, ionic moiety, buffer composition and the mode of migration of the polyion through the gel matrix.
  • biopolymers migrate under the influence of an electric field by tumbling through pores whose average radii are much larger that the radius of gyration of the analyte. Migrating samples are thereby size-ordered based on the time required to find a path through the pores of the gel matrix.
  • This type of migration is known as separation in the Ogston regime, and is usually quite time-consuming. Larger molecules, i.e. those molecules whose radii of gyration are larger than the average pore size, are impeded and become oriented towards the electric field while migrating through the pores. This process can be induced through increases in either the gel concentration or the applied electric field strength.
  • Non-crosslinked polymers may be supplied in a dessicated dry form, thereby providing a practically unlimited shelf life. Planar non-crosslinked polymer gels can be easily re- hydrated to any final gel concentration, buffer composition or strength.
  • the present invention is directed to a three-dimensional SDS PAGE technique for the separation of proteins refened to herein as 3-D sodium dodecyl sulfate-polyacrylamide cube gel electrophoresis (SDS PACGE).
  • the first dimension involves the separation of proteins by isoelectric point (pi)
  • the second dimension involves the separation of the focused proteins from the first dimension by molecular weight with a high percentage SDS-PAGE
  • the third dimension employs a second SDS-PAGE separation method by which the high molecular with proteins are separated by molecular weight with a low percentage SDS-PACGE.
  • the utilization of two SDS-PAGE steps in 3-D SDS-PACGE compared to one single step in 2-D SDS-PAGE has increased the separation between proteins in a single analysis.
  • the high percentage SDS-PAGE enhances the separation of the low molecular weight proteins.
  • the low percentage SDS-PAGE enhances the separation of the high molecular weight proteins.
  • in one or more of the dimensions of separation the proteins are separated according to given functional characteristics.
  • the present disclosure is directed to a three- dimensional electrophoresis apparatus for the separation of the components of a mixture which comprises a three dimensional separation medium in which the components of the material mixture to be separated are at least partially spatially resolved according to one or more characteristics of the components of the mixture by migration under the influence of an electrical field along a first dimension of the separation medium; the partially spatially resolved by migration along the first dimension are further spatially resolved by migration under the influence of an electrical field along a second dimension of the separation medium under conditions different from those pertaining to migration along the first dimension; and the components of the material mixture to be separated that have been at least partially spatially resolved by migration along the first and second dimensions are yet further spatially resolved by migration under the influence of an electrical field along a third dimension of the separation medium under conditions different from those pertaining to migration along the first and second dimensions.
  • the apparatus preferably contains a source for supplying an electrical field across opposed faces of the first, second or third dimension of the separation medium in accord with the dimension along which separation of the components of the mixture is presently being performed.
  • the apparatus will be provided with a source that supplies electrical potentials and cunents suitable for establishing the desired electrical fields across opposed faces of the separation medium.
  • the apparatus also may contain a mode for the in-situ detection of the spatially resolved material mixture wherein said the apparatus contains a detector positioned adjacent to the third dimension of the separation medium.
  • the separation media of the first, and/or second, and/or third dimensions comprises an anticonvective separation medium selected from the group consisting of a gelatinous crosslinked polymer, a solution of one or more non-crosslinked linear polymers, a suspension of one or more non-crosslinked linear polymers and a porous membrane.
  • the anticonvective separation medium of the first, second and third dimensions comprises a crosslinked acrylamide — methylene-bis-acrylamide copolymer gel.
  • the anticonvective separation medium of the first, second and third dimensions comprises an agarose gel.
  • the apparatus of the invention is used in a method in which the component mixture to be resolved is initially applied to one or more locations on the first dimension of the separation medium.
  • the apparatus may be used in a method in which the component mixture to be resolved is incorporated into the medium of the first dimension.
  • the apparatus of the present invention preferably employs a pH gradient in the first dimension.
  • the pH gradient is established between two opposed faces of the first dimension of separation, and the pH gradient resolves the components of the material mixture to the separated on the basis of the isoelectric points of the individual components.
  • a pH gradient is established by use of canier ampholytes.
  • the pH gradient is immobilized within the medium of the first dimension.
  • Such prefened embodiments may be further characterized in that the first dimension is in the form of an immobilized pH gradient (IPG) gel.
  • IPG immobilized pH gradient
  • the three-dimensional electrophoresis apparatus of the invention is used in methods in which the spatial resolution of the components of the material mixture occurs in the second and third dimensions on the basis of the hydrodynamic radii of the components wherein the media of the second and third dimensions differ in the resistance offered to the migration of components of the mixture each component having a different hydrodynamic radius within some range of radii.
  • separation is exemplified by PAGE, and more specifically SDS PAGE.
  • the three-dimensional electrophoresis apparatus of invention is preferably one which is used in a method in which the spatial resolution of the components of the material mixture occurs in the on the basis of the different strengths of binding interactions between individual components of the mixture and the solid components of the second dimension of the separation medium More specifically, the solid components of the second dimension of the separation medium comprise structurally incorporated therein chemical entities that preferentially bind to a specific component or group of components of the mixture over other components of the mixture. Specific entities that may be used to confer functionality to the dimension of separation include antibodies specific for a given component in the mixture to be separated.
  • the relative resistances to the migration of components of the mixture in the second and third directions is determined and controlled by the concentrations of the monomer(s) and crosslinking agent(s) employed in the preparation of the separation media in said second and third directions.
  • the medium of the second dimension may comprise an acrylamide- methylene-bis-acrylamide gel containing between about 10% and 18% and preferably about 12% acrylamide.
  • the medium of the third dimension preferably comprises an acrylamide-methylene-bis-acrylamide gel containing between about 3% and 10% and preferably about 7.5% acrylamide.
  • the apparatus of the present invention is particularly contemplated for use in methods for the separation of materials in which the component mixture to be resolved is comprised of complexes of protein with the detergent sodium dodecylsulfate (SDS) formed under conditions where the charge to mass ratios of the complexes are nominally the same or similar and where the hydrodynamic radii of the complexes are a nominal function of the molecular weight of the protein forming the complex.
  • the apparatus of the present invention preferably comprises a detection device wherein the spatially resolved components are detected optically.
  • the detection device is such that it is able to detect optical contrast between resolved components and the separation medium.
  • Such a contrast may be created or enhanced by means of one or more staining dyes selected from among chromatic dyes, chromogenic dyes, fluorescent dyes, and fluorogenic dyes.
  • the mixture to be resolved may preferably be treated with one or more staining dyes prior to being resolved.
  • some or all of the components of the mixture to be resolved are exposed to, and bind or react with, a staining dye dispersed throughout the separation medium during the course of resolution.
  • the mixture components resolved in a second dimension are preferably treated with a staining dye before being resolved in a third dimension.
  • Particularly prefened staining dyes include but are not limited to Procion and Ramazol dyes; textile industry Diazo chloromercury reactive dyes, and naphthoic disulfide dyes which react with cysteine. More specifically, the dyes include Uniblue A, Remazol Brilliant Violet, Reactive Blue 4, Reactive Blue 5, Reactive Blue 2, Reactive Orange 16, Reactive Orange 14, Reactive yellow 86, Reactive Green 5, Reactive Green 19, Reactive Brown 10, Reactive Red 120, Procion yellow H-E3G.
  • the detectors used in the apparatus of the invention include for example, fiber optics-based, laser-induced fluorescence systems.
  • the detector is a Polaroid and/or a video monitor.
  • the apparatus of the invention preferably comprises a detector that comprises a source that emits light at a wavelength or within a wavelength band that is absorbed by the staining dye or dyes; a lens set for focusing light from the source onto the detection area; and a charge coupled device (CCD) based hyper-spectral image capture and analysis system.
  • a detector that comprises a source that emits light at a wavelength or within a wavelength band that is absorbed by the staining dye or dyes; a lens set for focusing light from the source onto the detection area; and a charge coupled device (CCD) based hyper-spectral image capture and analysis system.
  • CCD charge coupled device
  • the three-dimensional electrophoresis , apparatus is one in which the resolution of the components of a mixture in a first dimension is performed within a discrete linear separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution in a second and a third dimension is performed.
  • the apparatus is one in which the resolution of the components of a mixture in a first and a second dimension is performed within a discrete planar separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution on a third dimension is performed.
  • the present invention further contemplates a method for spatially resolving a mixture of proteins within an anisotropic separation medium, wherein the proteins are resolved by migration under the influence of an electrical field along a first dimension of the separation medium, said migration being modulated by the characteristics of the separation medium in that dimension; the proteins partially resolved by migration along the first dimension of the separation medium are then further resolved by migration under the influence of an electrical field along a second dimension of the separation medium, the migration along the second dimension being modulated by characteristics of the separation medium in the second dimension that differ from the characteristics in the first dimension; the proteins partially resolved by migration along a first and a second dimension of the separation medium are then further resolved by migration under the influence of an electrical field along a third dimension of the separation medium, the migration along the third dimension being modulated by characteristics of the separation medium in the third dimension that differ from the characteristics in the first and second dimensions; and detecting the spatial locations of the resulting protein zones within the three-dimensional volume of the separation medium.
  • the resolution along the first dimension of the separation medium is on the basis of the isoelectric points of the proteins comprising the mixture.
  • the method of the invention comprises performing a resolution along the second dimension of the separation medium which is performed on the basis of the hydrodynamic radii of the proteins comprising the mixture, the characteristics of the separation medium in said second dimension significantly restrict the migration of the proteins within the mixture that have large hydrodynamic radii relative to those of other proteins within the mixture.
  • the resolution along the third dimension of the separation medium is on the basis of the hydrodynamic radii of the proteins comprising the mixture, the characteristics of the separation medium in said third dimension being less restrictive to the migration of the proteins within the mixture that have large hydrodynamic radii than is the separation medium in the second dimension.
  • the method is one which is used to resolve a protein mixture that is comprised of complexes of the proteins with the detergent sodium dodecylsulfate (SDS) formed under conditions where the charge to mass ratios of the complexes are nominally the same or similar and where the hydrodynamic radii of the complexes are a nominal function of the molecular weights of the proteins forming complexes.
  • SDS detergent sodium dodecylsulfate
  • proteins within the mixture being resolved specifically bind to a moiety that is incorporated into the structure of the second dimension of the separation medium.
  • the detection of the resolved proteins preferably comprises staining the proteins with one or more staining dyes, illuminating the stained proteins with light of one or more wavelengths that are absorbed by the staining dyes and optically visualizing the stained proteins.
  • the staining dyes are chromatic dyes typically employed in the visualizing of proteins and detection comprises the measurement of the diminution of the illuminating light intensity resulting from the absorbance of the light by staining dye attached to the proteins.
  • the staining dyes are fluorescent and detection comprises the measurement of the fluorescent light emitted by staining dye(s) attached to the proteins.
  • the staining dye is dispersed throughout one or more of the dimensions of the separation medium.
  • the dye is preferably dispersed through the second dimension of the separation medium and it binds to the proteins as they are resolved by migration through said second dimension of the separation medium.
  • the proteins are treated with the staining dye before being subjected to resolution in the third dimensions.
  • FIG. 1 Apparatus of three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE).
  • A Cube gel cell with internal dimensions of 3 ⁇ "(l) x 3 y 8 "(w) x 3 W'Qa) with a thickness of %".
  • B Cube gel bottom tray with internal dimensions of 3 5 /s"(l) x 3 5 /s"(w) x 1 ⁇ "(h) with a thickness of V”.
  • C The assembly of the 3-D SDS-PACGE apparatus.
  • D The copper screen cathode of the 3-D SDS-PACGE with dimensions of 3" x 3" with 15 x 15 lines per sq. in. and a wire diameter of 0.01 in.
  • E The bottom cube gel cell holder pieces with dimensions of 3 "(1) x 3 t"(w) x A"( ). Cube was run at 10 watts constant.
  • Figure 2 (A) 12% NuPAGE® Bis-Tris 1-D SDS-PAGE with MES running buffer, (B) 7.5% Tris-Glycine 1-D SDS-PAGE, and (C) side view of 3-D SDS-PACGE of the wide range multi-colored protein Mw standard.
  • the standard contains myosin (rabbit muscle, 205 kDa, iris blue), phosphorylase B ( rabbit muscle, 111 kDa, outrigger orange), glutamic dehydrogenase (bovine liver, 52 kDa, magenta), carbonic anhydrase (bovine erythrocytes, 34 kDa, hi-gloss Phoenician purple), myoglobin blue (horse heart, 19 kDa, flat Venetian blue), myoglobin red (horse heart, 17 kDa, pink), lysozyme (chicken egg white, 11 kDa, slicker yellow), aprotinin( bovine milk, 6,000 Da, pink), and insulin( bovine, 3,000 Da, hi-gloss true blue).
  • FIG. 3 (A) Bird's-eye view of the 3-D SDS-PACGE and (B) side view of the 3-D SDS-PACGE of bovine serum albumin.
  • First dimension LPG 3-10; separation distance, 7 cm.
  • Second dimension vertical 12% NuPAGE® Bis-Tris SDS- PAGE; running buffer, MES.
  • Third dimension 7.5% Tris-Glycine SDS-PACGE; cube gel dimension, 3 ⁇ "(l) x 3 ! g"(w) x 2 ⁇ "(h). Arrows point to the higher Mw and higher pi values.
  • FIG. 4 Separation of biotinylated insulin (20 ⁇ g) and biotinylated protein A (20 ⁇ g) by functionality using a 5% Tris-Glycine native polyacrylamide gel.
  • An arrow indicates a 15 % trapping gel piece.
  • Lane 1 shows a control gel which does not have a 15 % trapping gel piece;
  • lane 2 has a 15 % trapping gel piece with Human IgG (100 ⁇ g) as the trapping agent;
  • lane 3 has a 15 % trapping gel piece with Avidin (100 ⁇ g) as the trapping agent;
  • lane 4 has a 15 % trapping gel piece without any protein trapping agent.
  • 2-D SDS PAGE has been known for many years as a versatile technique for the separation of proteins.
  • 2-D SDS PAGE is ineffective at producing adequate and efficient resolution of the protein mixture.
  • the present invention describes methods and compositions for a three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE) technique which overcomes the prior cumbersome methods and provides better protein resolution than is seen in 2-D SDS PAGE.
  • the present invention for the first time provides details of an enhanced method for the separation of proteins in a single analysis using SDS-PAGE.
  • the improved resolution seen in the present invention is achieved by introducing a third dimension in addition to 2-D SDS-PAGE.
  • the first dimension focuses the proteins of a mixture using IEF, much like the first step in 2-D SDS-PAGE.
  • the focused proteins are then subjected to a separation in a second dimension in which the SDS-PAGE is performed to achieve resolution of low Mw proteins in the mixture.
  • the SDS-PAGE in the second dimension is a high percentage (e.g., 12 %) SDS-PAGE with 2-[N-Morpholino]ethanesulfonic acid (MES) running buffer.
  • the third dimension of the 3-D SDS-PACGE involves subjecting the gel containing the separated proteins from the second dimension to a second SDS-PAGE step in order to achieve resolution of high Mw proteins in the protein mixture. This third dimension is preferably carried out in a low percentage (e.g., 7.5 %) SDS-PAGE cube gel with Tris-Glycine gel running buffer.
  • the utilization of two SDS-PAGE steps in 3-D SDS-PACGE compared to one single step in 2-D SDS-PAGE has increased the separation between proteins in a single analysis.
  • the high percentage SDS-PAGE enhances the separation of the low molecular weight proteins.
  • the low percentage SDS-PAGE enhances the separation of the high molecular weight proteins. While prefened embodiments of the present application detail that the second dimension separation step is a high percentage SDS PAGE and the third dimension is a low percentage SDS PAGE separation, it should be understood that the order of these steps may be reversed.
  • FIG. 1 A The cube gel was made by a cell ( Figure 1 A) of acrylic with a thickness of 0.25 inches and internal dimensions of 3.5 inches (length) x 3.5 inches (width) x 3.5 inches (height).
  • Figure 1 A A copper screen (3 inches x 3 inches, 15 15 lines per square inch with a wire diameter of 0.01 in.) soldered to a copper wire was used as the cathode ( Figure ID) of the 3-D SDS-PACGE.
  • Figure IB shows the cube gel bottom tray with a thickness of 0.25 inches and internal dimensions of 3.625 inches (1) x 3.625 inches (width) x 1.25 inches (height) for holding the lower buffer.
  • the anode of the 3-D SDS-PACGE is an 18 inch long platinum wire running the perimeter of the cube gel cell.
  • PowerEase ® 500 programmable power supply (Invitrogen, Carlsbad, CA) was used as the power supply.
  • the whole 3-D SDS-PACGE apparatus may be covered by a plastic box during the run for safety.
  • the cube gel of the 3-D SDS-PACGE was prepared as follows: A 7.5% polyacrylamide resolving gel solution (400 ml) was poured into the cube gel cell, which was sealed by a plastic plate on the bottom with universal box sealing tape, and allowed to polymerize.
  • the 7.5% polyacrylamide resolving gel solution was prepared by mixing 100 ml 30.8% stock monomer acrylamide solution, 100 ml of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 4 ml of 10% SDS, 194 ml of ddH2O, 2 ml of APS (freshly prepared), and 133 ⁇ l of TEMED. Following gel formation, a 4% polyacrylamide stacking gel solution
  • the 4% polyacrylamide stacking gel solution was prepared by mixing 8 ml stock monomer acrylamide solution, 15 ml of 0.5 M Tris-HCl stacking gel buffer (pH 6.8), 0.6 ml of 10% SDS, 36 ml of ddH2O, 300 ⁇ l of APS (freshly prepared), and 30 ul of TEMED. Since air bubbles inside the cube gel are detrimental to the running of the 3-D SDS- PACGE, vacuum was applied to both resolving and stacking gel solutions while stirring on a magnetic stiner for several minutes to deaerate the solution.
  • the sealing plastic plate was removed from the bottom and the cube gel cell was placed in the center of the bottom tray, and supported by the two supporting pieces.
  • Fifty ml of gel running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) was added to the top of the cube gel cell (upper buffer reservoir) and 200 ml of gel running buffer was added to the bottom tray (lower buffer reservoir).
  • the gel was laid on top of the cube gel (3.125 inches (length) x 3.125 inches (width) x 2.5 inches (h)). Electrophoresis was carried out at 10 watts with a cunent of ⁇ 250 mA and a voltage of approximately 40 volts for approximately 4 hours.
  • the copper screen is used instead of copper plate as the cathode to prevent accumulation of air bubbles which will decrease the efficiency of the apparatus.
  • Running the 3-D SDS-PACGE at higher wattage caused the cube gel to expand and crack due to a rise in gel temperature.
  • BSA 100 ⁇ g in the 2-D SDS- PAGE gel were stained covalently with 20 ml freshly prepared Remazol Brilliant Violet 5R (10 mg/ml) at room temperature for 30 min. at pH 11 in 10 mM CAPS buffer and then de-stained in water for 15 min. This staining process was repeated three times before running the cube gel.
  • the 2-D SDS-PAGE gels were stained covalently with 20 ml freshly prepared Uniblue A (5mg/ml) in water at room temperature for 30 min. before staining with 20 ml freshly prepared Remazol Brilliant Violet 5R (10 mg/ml) at room temperature for 30 min at pH 11 in 10 mM CAPS buffer.
  • U.S. Patent No. 6,507,664 describes two-dimensional gels for use in electrophoresis methods
  • U.S Patent No. 6,398,933 describes a two dimensional electrophoresis system
  • U.S. Patent Nos 6,482,303; 6,480,618; 6,451,189 describes an automated system for use in two- dimensional electrophoresis
  • microchip devices for use in the operation and control of electrophoresis systems are described in U.S. Patent No. 6,319,705
  • protein sample preparation for electrophoresis is described in U.S. Patent No. 6,391,650
  • additional methods and sample preparations for 2D electrophoresis are described in U.S. Patent Nos.
  • the second dimension may be modified such that proteins are separated according to given functional characteristics.
  • the dimension of the "functionality" will preferably be the second dimension, however, it is contemplated that the third or even the first dimension may be the dimension on which the mixture is resolved according to functionality.
  • This functionality dimension will be used to separate the complex protein mixture according to different functionalities such as glycoprotein, lipoprotein, phosphorylated protein, antibodies, and the like.
  • Such separation may be achieved by preparing the matrix of the second dimension (e.g., low percentage native PAGE gel) with many layers of affinity tags that are specific for the given class of proteins to be separated.
  • the affinity tags may be trapped in the native PAGE gel or covalently linked the gel matrix.
  • Exemplary affinity tags for use in the second dimension include lectin for the separation of glycoproteins, antibodies against phosphorylated proteins, lipoproteins and the like.
  • this 3D separation may comprise in the first dimension a conventional IEF separation matrix in order to separate proteins by PI.
  • the second dimension is one designed specifically to receive and further separate proteins, which were separated in the first dimension, according to the functionality of the proteins in the complex mixture and the third dimension comprises an SDS PAGE matrix to effectuate the separation of proteins separated in the second, dimension according to molecular weight.
  • the matrix for the second dimension may be polyacrylamide gel, agarose, a mixture of polyacrylamide and agarose or any other matrix to facilitate the separation of proteins.
  • the matrix for the second dimension is a polyacrylamide gel native gel without SDS.
  • Agents that will bind to the proteins of interest e.g., substrates or affinity tags for the proteins are trapped in a high percentage area of the gel using conventional polyacrylamide gel preparation techniques well known to those of skill in the art. The remainder of the gel is a low percentage gel so protein can pass freely. While the proteins are passing through this area, only proteins that bind with the substrates with be retained as they bind to the affinity tags.
  • the affinity tags are placed into a solution before the acrylamide is polymerized. Substrates can be trapped covalently by copolymerization with the acrylamide.
  • the tag is labeled with acrylic acid n-hydroxy- succinimide ester and copolymerized with acrylamide.
  • this "functional separation" dimension is set up as follows.
  • about 10 ml of the resolving gel solution was poured into a 10 x 10 cm empty cassette with a thickness of 1 mm (Invitrogen).
  • a comb was inserted on the top of the cassette to wells.
  • the 5% polyacrylamide resolving gel solution was prepared by mixing 2.5 ml 30.8% stock monomer acrylamide solution, 3.75 ml of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 8.7 ml of ddH2O, 75 ⁇ l of APS (freshly prepared), and 5 ⁇ l of TEMED. Following gel formation and comb removed, 30 ⁇ l of 15% trapping gel solution which contains no protein, Avidin, or Human IgG was loaded on top of each well.
  • Trapping gel solution was prepared by mixing 15 ⁇ l stock monomer acrylamide solution, 7.5 ⁇ l of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 7.4 ⁇ l of ddH2O, 1 ⁇ l of APS (freshly prepared), and 0.1 ⁇ l of TEMED.
  • the trapping moieties used to retain the proteins are avidin and human IgG, however, it should be understood that those of skill will be able to modify the separation technique by using a trapping moiety other than avidin, e.g., antibodies specific for phosphorylation status, glycoprotein-trapping moieties, ligands for receptors etc.
  • a protein is a macromolecule composed of a chain of amino acids. Of the 20 amino acids found in typical proteins, four (aspartic and glutamic acids, cysteine and tyrosine) cany a negative charge and three (lysine, arginine and histidine) a positive charge, in some pH range.
  • a specific protein defined by its specific sequence of amino acids, is thus likely to incorporate a number of charged groups along its length.
  • the magnitude of the charge contributed by each amino acid is governed by the prevailing pH of the sunounding solution, and can vary from a minimum of 0 to a maximum of 1 charge (positive or negative depending on the amino acid), according to a titration curve relating charge and pH according to the pK of the amino acid in question. Under denaturing conditions in which all of the amino acids are exposed, the total charge of the protein molecule is given approximately by the sum of the charges of its component amino acids, all at the prevailing solution pH.
  • Two proteins having different ratios of charged, or titrating, amino acids can be separated by virtue of their different net charges at some pH. Under the influence of an applied electric field, a more highly charged protein will move faster than a less highly charged protein of similar size and shape. If the proteins are made to move from a sample zone through a non-convecting medium (typically a gel such as polyacrylamide), an electrophoretic separation will result. If, in the course of migrating under an applied electric field, a protein enters a region whose pH has that value at which the protein's net charge is zero (the isoelectric pH), it will cease to migrate relative to the medium. Further, if the migration occurs through a monotonic pH gradient, the protein will "focus" at this isoelectric pH value.
  • a non-convecting medium typically a gel such as polyacrylamide
  • isoelectric focusing can resolve two proteins differing by less than a single charged amino acid among hundreds in the respective sequences.
  • a key requirement for an isoelectric focusing procedure is the formation of an appropriate spatial pH gradient. This can be achieved either dynamically, by including a heterogeneous mixture of charged molecules (ampholytes) into an initially homogeneous separation medium, or statically, by incorporating a spatial gradient of titrating groups into the gel matrix through which the migration will occur.
  • the former represents classical ampholyte-based isoelectric focusing, and the latter the more recently developed immobilized pH gradient (LPG) isoelectric focusing technique.
  • LPG approach has the advantage that the pH gradient is fixed in the gel, while the ampholyte-based approach is susceptible to positional drift as the ampholyte molecules move in the applied electric field.
  • the best cunent methodology combines the two approaches to provide a system where the pH gradient is spatially fixed but small amounts of ampholytes are present to decrease the adsorption of proteins onto the charged gel matrix of the LPG
  • IPG gels in a thin planar configuration bonded to an inert substrate, typically a sheet of Mylar plastic which has been treated so as to chemically bond to an acrylamide gel (e.g., Gelbond® PAG film, FMC Corporation).
  • the LPG gel is typically formed as a rectangular plate 0.5 mm thick, 10 to 30 cm long (in the direction of separation) and about 10 cm wide. Multiple samples can be applied to such a gel in parallel lanes, with the attendant problem of diffusion of proteins between lanes producing cross contamination.
  • sample entry area is typically smaller than the gel surface forming the well bottom because the protein migrates into the gel under the influence of an electric field which directs most of it to one edge of the well bottom, tending to produce protein precipitation.
  • the major source of precipitation is provided by the charged groups introduced into the gel matrix to form the pH gradient in IPG gels: these groups can interact with charges on the proteins (most of which are not at their isoelectric points at the position of the application point and hence have non-zero net charges) to bind precipitates to the gel.
  • LPG immobilized pH gradient
  • sample wells used for the application of macromolecular-containing samples to the surfaces of gels, most frequently slab gels used for protein or nucleic acid separations.
  • sample wells are generally designed to concentrate macromolecules in the sample into a thin starting zone prior to their migration through the resolving gel.
  • U.S. Patent No. 5,304,292 describes the use of pieces of compressible gasket to form well walls at the top of a slab where the ends of the pieces touch the top edge of the slab.
  • U.S. Patent No. 5,164,065 describes a shark's tooth comb used in combination with DNA sequencing gels.
  • U.S. Patent No. 5,074,981 discloses a substitute for submarine gels using an agarose block that is thicker at the ends and hangs into buffer reservoirs.
  • U.S. Patent No. 5,275,710 discloses lane-shaped gels formed in a plate and gel- filled holes extending down from the plate into buffer reservoirs. Any of the methods for preparing IPG gels described in these patents listed in the present section may be adapted for the SDS PACGE methods of the present invention.
  • the second and third dimensions of the SDS PACGE methods described herein employ PAGE based methods.
  • charged detergents such as sodium dodecyl sulfate (SDS) can bind strongly to protein molecules and "unfold" them into semi-rigid rods whose lengths are proportional to the length of the polypeptide chain, and hence approximately proportional to molecular weight.
  • SDS-PACGE charged detergents
  • the methods of the present invention are directed to SDS-PACGE in which the inert matrix is polyacrylamide. It should be understood that those of skill in the art may adapt the methods to the use of other polymers including but not limited to non-cross linked polyacrylamide, dextran, polyethylene oxides, derivatized celluloses, polyvinylpynolidone and mixtures thereof.
  • a protein complexed with such a detergent is itself highly charged (because of the charges of the bound detergent molecules), and this charge causes the protein-detergent complex to move in an applied electric field. Furthermore, the total charge also is approximately proportional to molecular weight (since the detergent's charge vastly exceeds the protein's own intrinsic charge), and hence the charge per unit length of a protein-SDS complex is essentially independent of molecular weight.
  • This feature gives protein-SDS complexes essentially equal electrophoretic mobility in a non-restrictive medium. If the migration occurs in a sieving medium, such as a polyacrylamide gel, however, large (long) molecules will be retarded compared to small (short) molecules, and a separation based approximately on molecular weight will be achieved. This is the principle of SDS electrophoresis as applied commonly to the analytical separation of proteins.
  • SDS electrophoresis involves the use of a slab-shaped electrophoresis gel as the second dimension of a two-dimensional procedure.
  • the gel strip or cylinder in which the protein sample has been resolved by isoelectric focusing is placed along the slab gel edge and the molecules it contains are separated in the slab, perpendicular to the prior separation, to yield a two-dimensional (2-D) separation.
  • 2-D two-dimensional
  • the gel is molded by introducing a dissolved mixture of polymerizable monomers, catalyst and initiator into the cavity defined by the plates and spacers or gaskets sealing three sides. Polymerization of the monomers then produces the desired gel media. This process is typically carried out in a laboratory setting, in which a single individual prepares, loads and rans the gel. A gasket or form comprising the bottom of the molding cavity is removed after gel polymerization in order to allow cunent to pass through two opposite edges of the gel slab: one of these edges represents the open (top) surface of the gel cavity, and the other is formed against its removable bottom. Typically, the gel is removed from the cassette defined by the glass plates after the electrophoresis separation has taken place, for the purposes of staining, autoradiography, etc., required for detection of resolved macromolecules such as proteins.
  • %T the total percentage of acrylamide in the gel by weight
  • %C the proportion of the total acrylamide that is accounted for by the crosslinker used.
  • N,N'-methylenebisacrylamide (“bis") is typically used as crosslinker.
  • Typical gels used to resolve proteins range from 8% T to 24% T, a single gel often incorporating a gradient in order to resolve abroad range of protein molecular masses.
  • the second dimension separation preferably employs a high percentage SDS PAGE gel such as for example 12 % SDS-PAGE with 2-[N-Morpholino]ethanesulfonic acid (MES) running buffer to separate low Mw proteins.
  • MES 2-[N-Morpholino]ethanesulfonic acid
  • any SDS-PAGE gel having greater than SDS PAGE and preferably between about 10% to about 24% SDS PAGE may be used for the second dimension.
  • running buffers other than MES that are typically used in running high percentage SDS PAGE systems may be used and include but are not limited to Tris glycine, Tris Page and the like.
  • the third dimension separation in the methods of the present invention is designed to separate the high molecular weight proteins in a sample and employs a lower percentage SDS PAGE composition than in employed in the second dimension.
  • the third dimension employs a 7.5% SDS-PAGE cube gel with Tris-Glycine gel running buffer. Again the percentage of the SDS PAGE cube gel may be varied between 0.1% to about 10% percent SDS PAGE cube gel configuration.
  • the running buffer may be any buffer typically employed in running conventional low percentage SDS PAGE systems. Sambrook et al., supra, describes conditions for low and high molecular weight SDS PAGE systems at ⁇ A8.40 through A8.51.
  • percentage ranges and the linear range of separation of proteins on such gels is given in table A8.8 therein and indicates that a 15% acrylamide gel will likely yield a linear separation of proteins in the range of 10 kDa to about 43 kDa; a 12% acrylamide gel will likely yield a linear separation of proteins in the range of 12 kDa to about 60 kDa; a 10% acrylamide gel will likely yield a linear separation of proteins in the range of 20 kDa to about 80 kDa; a 7.5% acrylamide gel will likely yield a linear separation of proteins in the range of 36kDa to about 94 kDa; and a 5% acrylamide gel will likely yield a linear separation of proteins in the range of 57 kDa to about 212 kDa.
  • SDS electrophoresis use is made of the stacking phenomenon first employed in this context by Laemmli, U. K. (1970) Nature 227:680.
  • an additional gel phase of high porosity is interposed between the separating gel and the sample.
  • the two gels initially contain a different mobile ion from the ion source (typically a liquid buffer reservoir) above them: the gels contain chloride (a high mobility ion) and the buffer reservoir contains glycine (a lower mobility ion, whose mobility is pH dependent). All phases contain Tris as the low-mobility, pH determining other buffer component and positive counter-ion.
  • Negatively charged protein-SDS complexes present in the sample are electrophoresed first through the stacking gel at its pH of approximately 6.8, where the complexes have the same mobility as the boundary between the leading (C1-) and trailing (glycine-) ions.
  • the proteins are thus stacked into a very thin zone "sandwiched" between CI- and glycine-zones. As this stacking boundary reaches the top of the separating gel the proteins become unstac ed because, at the higher separating gel pH (8.6), the protein-SDS complexes have a lower mobility.
  • the proteins fall behind the stacking front and are separated from one another according to size as they migrate through the sieving environment of the lower porosity (higher %T acrylamide) separating gel. In this environment, proteins are resolved on the basis of mass.
  • Pre-made slab gels have been available commercially for many years (e.g., from Integrated Separation Systems). These gels are prepared in glass cassettes much as would be made in the user's laboratory, and shipped from a factory to the user. More recently, methods have been devised for manufacture of both slab gels in plastic cassettes (thereby decreasing the weight and fragility of the cassettes) and slab gels bonded to a plastic backing (e.g., bonded to a Gelbond® Mylar® sheet or to a suitably derivatized glass plate). To date, all commercially-prepared gels are either enclosed in a cassette or bonded to a plastic sheet on one surface (the other being covered by a removable plastic membrane). Furthermore, all commercially-prepared gels have a planar geometry. The methods and compositions employed to prepare these pre-made gels also may be used in the preparation of the 3D cubes for the present invention.
  • Cunent practice in running slab gels involves one of two methods.
  • a gel in a cassette is typically mounted on a suitable electrophoresis apparatus, so that one edge of the gel contacts a first buffer reservoir containing an electrode (typically a platinum wire) and the opposite gel edge contacts a second reservoir with a second electrode, steps being taken so that the cunent passing between the electrodes is confined to run mainly or exclusively through the gel.
  • Such apparatus may be "vertical" in that the gel's upper edge is in contact with an upper buffer reservoir and the lower edge is in contact with a lower reservoir, or the gel may be rotated 90° about an axis perpendicular to its plane, so that the gel runs horizontally between a left and right buffer reservoir, as is disclosed in U.S. Pat. No. 4,088,561 (e.g., "DALT" electrophoresis tank).
  • Various configurations have been devised in order to make these connections electrically, and to simultaneously prevent liquid leakage from one reservoir to the other (around the gel).
  • an IEF gel When used as part of a typical 2-D procedure, an IEF gel is applied along one exposed edge of such a slab gel and the proteins it contains migrate into the gel under the influence of an applied electric field.
  • the IEF gel may be equilibrated with solutions containing SDS, buffer and thiol reducing agents prior to placement on the SDS gel, in order to ensure that the proteins the LEF gel contains are prepared to begin migrating under optimal conditions, or else this equilibration may be performed in situ by sunounding the gel with a solution or gel containing these components after it has been placed in position along the slab's edge.
  • a slab gel affixed to a Gelbond® sheet is typically run in a horizontal position, lying flat on a horizontal cooling plate with the Gelbond® sheet down and the unbonded surface up. Electrode wicks communicating with liquid buffer reservoirs, or bars of buffer-containing gel, are placed on opposite edges of the slab to make electrical connections for the nm, and samples are generally applied onto the top surface of the slab (as in the instractions for the Pharmacia ExcelGels).
  • the proteins in 2-D gels generally are detected either by staining the gels or by exposing the gels to a radiosensitive film or plate (in the case of radioactively labeled proteins).
  • Staining methods include dye-binding (e.g. Coomassie Brilliant Blue), silver stains (in which silver grains are formed in protein- containing zones), negative stains in which, for example, SDS is precipitated by Zn ions in regions where protein is absent, or the proteins may be fluorescently labeled.
  • dye-binding e.g. Coomassie Brilliant Blue
  • silver stains in which silver grains are formed in protein- containing zones
  • negative stains in which, for example, SDS is precipitated by Zn ions in regions where protein is absent
  • the proteins may be fluorescently labeled.
  • images of separated protein spot patterns can be acquired by scanners, and this data reduced to provide positional and quantitative information on sample protein composition through the action of suitable computer software.
  • the dyes used to stain the proteins should preferably be those that for strong, e.g., covalent bonds with the proteins in the 3D cube.
  • Such dyes include but are not limited to, reactive dyes such as Procion and Ramazol dyes; textile industry Diazo chloromercury-type reactive dyes, dyes react with phenolic or naphthoic protein moiety, and naphthoic disulfide dyes which react with cystine may be used.
  • reactive dyes such as Procion and Ramazol dyes
  • Diazo chloromercury-type reactive dyes dyes react with phenolic or naphthoic protein moiety
  • naphthoic disulfide dyes which react with cystine may be used.
  • the bovine serum albumin and a broad range multi-colored protein Mw standard were used to provide evidence of the efficacy and usefulness of the 3-D SDS PACGE technique.
  • This Example demonstrates that a mixture of proteins of a broad range of molecular weights can be separated using the instant technique. Since multi-colored protein Mw standard obtained from companies contained SDS and chemicals that are not suitable for LEF ran, multi-colored protein standard was run directly on the 12% NuPAGE® Bis-Tris gel and then the cube gel without the IEF step. The bovine serum albumin was however used as a control sample that was subjected to all three steps.
  • FIG. 2C The separation of a broad range multi-colored protein Mw standard using the 3-D SDS-PACGE and 1-D SDS-PAGE are shown in Figure 2.
  • the side views of the cube gel ran ( Figures 2C) shows the 12 % SDS-PAGE (MES running buffer) dimension and the 7.5 % SDS-PAGE (Tris-Glycine ranning buffer) dimension. The IEF dimension was not used.
  • the conesponding 12% 1-D SDS- PAGE and 7.5% 1-D SDS-PAGE are shown in Figure 2A and Figure 2B, respectively.
  • a direct comparison between the cube gel and the 1-D SDS-PAGE gels can be made. All proteins are resolved with the conect molecular weights by the cube gel.
  • the cube gel exhibits resolution of the protein separation of both the 12 % SDS- PAGE and 7.5 % SDS-PAGE gels in a single experiment run.
  • the cube gel ran of the broad range protein standard showed that the separation between phosphorylase B and glutamic dehydrogenase is enhanced in the 7.5 % SDS-PAGE dimension because the pore size of the 7.5% SDS-PAGE gel is optimized to separate high Mw proteins. Further, the separations of the low Mw proteins myoglobin blue, myoglobin red, lysozyme, aprotinin, and insulin are enhanced with the 12 % SDS-PAGE dimension because the pore size of the 12% SDS-PAGE gel is optimized to separate low Mw proteins.
  • Figure 3 shows the 3-D SDS-PACGE results of BSA.
  • Figure 3A shows a bird's-eye view of the cube gel with pi 3-10 as the first dimension and 12% SDS-PAGE as the second dimension.
  • Figure 3B shows a side view of the cube gel with 12% SDS-PAGE as the first dimension and 7.5% SDS-PAGE as the second dimension.
  • the 3-D SDS-PACGE exhibits resolution of the protein separation of both the 12 % SDS-PAGE and the 7.5 % SDS-PAGE gels in a single experiment run.
  • Figure 3 clearly shows the enhancement of the separation of the BSA and the high Mw contamination bands in a single experiment ran.
  • the second dimension of separation may employs the "functionality" to separate the components of a given mixture according to different functionalities such as glycoprotein, lipoprotein, phosphorylated protein, antibodies, and the like.
  • separation may be achieved by preparing the matrix of the second dimension (e.g., low percentage native PAGE gel) with many layers of affinity tags that are specific for the given class of proteins to be separated.
  • the affinity tags may be trapped in the native PAGE gel or covalently linked the gel matrix.
  • the present Example provide one exemplary separation of proteins using avidin, anti-BSA antibodies and concanavalin A incorporated into a 7.5% native PAGE. Using such antibodies embedded in the PAGE matrix, it is possible to anest the antigens at the site where the antigen-specific antibody is embedded.
  • the binding buffer was 20 mM sodium phosphate, 3 M NaCl, pH 7.
  • High binding buffer 1.5 M glycine, 3 M NaCl, pH 8.9.
  • the unpurified antibody is filtered through a 0.45 ⁇ m filter or centrifuged immediately before it is applied to the column.
  • the flow rate was 1 ml/min and the collection tubes contained 60-100 ⁇ l of 1 M Tris-HCl, pH 9 per ml of fraction to be collected.
  • the syringe or pump tubing is filled with starting buffer and the column is equilibrated with 5-10 column volumes of binding buffer.
  • the sample to be separated is applied to the column using a syringe fitted to the luer adaptor or by pumping the sample onto the column.
  • the column is then washed with 5 column volumes of binding buffer.
  • the antibody is eluted with 2- 5 column volumes of elution buffer.
  • the column is then washed five times with 20% ethanol to prevent microbial growth.
  • the purified IgG fractions can be desalted by buffer exchange using a PD-10 desalting column.
  • the 1-D polyacrylamide electrophoresis were prepared and run according to the manufacturer's (Invitrogen, Pharmacia) protocols.
  • the functional affinity electrophoresis (FAEP) gel was prepared as follows: A 7.5% polyacrylamide resolving gel solution (8 ml) was poured into a empty gel cassette (Invitrogen) with plastic strips positioned at a depth of 1/3 from the top of the gel to form the wells. The plastic strips have dimensions of 100 mm (1) x 8 mm (w) x 10 mm (h).
  • the 7.5% polyacrylamide resolving gel solution was prepared by mixing 7.5 ml 30.8% stock monomer acrylamide solution (Sigma), 7.5 ml of 1.5 M Tris-HCl 4 x resolving gel buffer (pH 8.8), 12.9 ml of ddH2O, 150 ⁇ l of 10% APS (ammonium persulfate solution), and 10 ⁇ l of TEMED (tetramethylethylenediamine). Following gel formation and removal of the plastic strips, 50 ⁇ l of the 7.5% polyacrylamide gel solution with or without 200 ⁇ g of avidin was added to the wells and allowed to polymerize. 75 ⁇ l of 7.5% polyacrylamide gel was poured and allow to polymerized.
  • the FAEP was performed to separate biotinylated insulin, BSA, and ovalbumin. These proteins were chosen because they carried negative charges while avidin carries a positive charge at the pH of the gel running buffer - 8.3.
  • the ID SDS-PAGE was used to detect the components of the avidin-biotinylated protein complex.
  • the complex was excised from the 5% CCAE gel and soaked in the SDS sample loading buffer to dissociate the avidimbiotinylated protein complex and the gel piece was loaded on the 4-12% SDS-NuPage gel (Invitrogen) with 2-[N- Morpholinojethanesulfonic acid (MES) running buffer.
  • Pierce' s TriChroRanger molecular weight marker was used to provide a MW calibration.
  • the complex was excised and subjected to trypsin in-gel digestion.
  • the protein gel was washed with 50% acetonitrile in 50 mM ammonium bicarbonate pH 8.0 and was shrunk with neat acetonitrile. The shrunk gel was then dried in speedvac. Reduction of the disulfide bond and alkylation of the free sulfhydryl group were done simultaneously by DTT (dithiothreitol) and 4-vinylpyridine in 6 M guanidine HCI 50 mM ammonium bicarbonate pH 8.0 with 5 mM EDTA , respectively.
  • Affinity electrophoresis has been used successfully for years in glycoprotein studies (Shariff and Parija, J. Microbiol. Methods 14 (1991) 71-761; Kurata and Tan, Arthritits Rheum 19 (1976) 574-580).
  • the sugar is covalently bind to the polyacrylamide and either pack in the gel or make a gel which can retard the movement of the analyte and allow the calculation of the binding constant.
  • the interaction force is not strong enough to counteract the electrophoresis force of migration.
  • Usually, what had been observed is a retardation of the migration and from this retardation kinetic parameters can be calculated. It is hard to observe the effect during the electrophoresis migration and it is dependent on the strength of the interaction.
  • Affinity electrophoresis have been used successfully to retard or even stop glycoproteins, plasma proteins, enzymes, nucleic acids, ⁇ ectins, receptors, and extracellular matrix proteins by the specific interactions with their ligands during electrophoretic migration in support media with a little molecular sieving effect (Shariff and Parija, J. Microbiol Methods 14 (1991) 71-761; Kurata and Tan, Arthritits Rheum 19 (1976) 574-580).
  • the FAEP method described above was used to separate proteins by their functions based on the principles of affinity electrophoresis and electroimmunodiffusion.
  • the avidin :biotin, con A:ovalbumin, and anti-BSA antibody:BSA complexes were used as a model system.
  • the avidin, anti-BSA, and con A are incorporated into separate layers of a 7.5% native PAGE gel.
  • Avidin 66 kDa
  • pi basic isoelectric point
  • Anti-BSA PI 8.4, MW 150 kDa
  • con A PI 5.1, MW 80 kDa
  • Biotinylated insulin bovine pancreas, PI 5.3, 6 kDa, 1 mole biotin per mole insulin
  • biotinylated bovine serum albumin BSA, 66 kDa, pi 5.5, 8-16 moles biotin per mole BSA
  • ovalbumin chicken, 45 kDa, pi 5.1
  • myoglobin myoglobin
  • BSA stopped at the anti-BSA ' antibody zone.
  • Mass spectrometry and sodium dodecyl sulfate polyacrymide (SDS- PAGE) were used to further confirm the formation of avidin :biotin, bsa:antiBSA (bonding constant), ovalbuminxon A (bonding constant) complexes.
  • peptide fragments at 2698 Da and 2924 Da were observed and were annotated as being from biotinylated insulin. Peaks at 1228 Da, 1440 Da, 1567 Da, 1663 Da, and 1774 Da were from biotinylated BSA, and a peak at 819 Da is from avidin after trypsin digestion of the protein complex gel. These results indicate that the complex was composed of avidin, biotinylated insulin, and biotinylated BSA. Interestingly, the peak at 2924 Da is the biotinylated peptide of 2698 Da of amino acid residue 1 to 21 of the biotinylated insulin chain B (a mass difference of 226). Peaks at 1228 Da, 1470 Da, 1663 Da, and many others are the biotinylated peptides of biotinylated BSA.
  • Example 2 The methods described in Example 2 were modified with a different set of functionalities to effect separation of a mixture of proteins.
  • Anti-ubiquitin, anti-BSA, and anti-GST antibodies were incorporated into a 7.5% native PAGE. Under gel running conditions, these antibodies do not move much compare to the analytes. The results of this separation showed that ubiquitin stops migrating in the gel when it encounters anti-ubiquitin, BSA stops migrating when it encounters anti-BSA antibody, and GST stops its migrating upon encountering anti- GST.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne des procédés et des compositions utilisés pour la technique d'électrophorèse sur gel de cube de sodium dodécyl sulfate-polyacrylamide tridimensionnel (3-D SDS-PACGE) afin d'augmenter la résolution de la séparation protéique. La technologie tridimensionnelle comprend la séparation de protéines par focalisation isoélectrique dans la première dimension, la séparation de poids moléculaire au moyen d'une SDS-PACGE à pourcentage élevé dans la seconde dimension, et la séparation de poids moléculaire au moyen d'une SDS-PACGE à pourcentage faible dans la troisième dimension. Dans des modes de réalisation spécifiques, un ou plusieurs procédés de l'invention peuvent employer un agent de piégeage qui confère à la dimension de séparation une fonctionnalité de sorte que des composantes spécifiques du mélange sont reliées à l'agent de piégeage au cours de la séparation. L'invention a également pour objet des procédés, des dispositifs et des compositions pour mettre en oeuvre la SDS-PACGE 3D.
PCT/US2004/002953 2003-02-06 2004-02-03 Procedes et compositions pour electrophorese sur gel de cube de sodium dodecyl sulfate-polyacrylamide 3-d WO2004072236A2 (fr)

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