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US 6,537,432 B1Granted
Protein separation via multidimensional electrophoresis
AssigneeTarget Discovery, Inc.
InventorsLuke V. Schneider, Michael P. Hall, Robert Petesch
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Abstract
The present method provides methods and apparatus for separating proteins using a series of electrophoretic methods that utilize controlled fractionation and labeling techniques to resolve mixtures of proteins. The samples for each electrophoretic method other than the initial method, contain only a subset of proteins resolved in the preceding method. The methods can be used in a variety of different applications including, creating proteomic databases, comparative expression studies, diagnostics, structure activity relationships and metabolic engineering investigations.
Claims55
A method for separating a plurality of proteins in an initial sample, comprising
- (a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, and wherein
- at least one of the plurality of methods is capillary isoelectric focusing electrophoresis (CIEF) or capillary zone electrophoresis (CZE) and the at least one method is conducted under conditions such that electroosmotic flow is less than or equal to 0.5×10−6 cm2/V-s;
- and
- (b) detecting resolved proteins from the final electrophoretic method.
1 more element
A method for separating a plurality of proteins in an initial sample, comprising
- (a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, whereby a plurality of resolved proteins are obtained;
- (b) detecting resolved proteins from the final electrophoretic method;
- (c) repeating steps (a) and (b) under the same conditions, wherein the initial sample for the first iteration of the method is from a first source and the initial sample for the second iteration of the method is from a second source;
- and
- (d) comparing the relative abundance of one or more of the resolved proteins obtained from the first source and the second source as detected from the final electrophoretic method.
2 more elements
A method for separating a plurality of proteins, comprising
- (a) performing a plurality of electrophoretic methods in series, wherein the method or methods preceding the final method comprise withdrawing and collecting multiple fractions containing proteins resolved during the electrophoretic method, and wherein each electrophoretic method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins and the final electrophoretic method which is conducted with a sample from one or more fractions obtained in the penultimate electrophoretic method;
- (b) labeling the plurality of proteins or labeling protein contained in one or more collected fractions prior to conducting the last electrophoretic method;
- and
- (c) detecting one or more labeled, resolved proteins obtained following the final electrophoretic method.
1 more element
A method for separating a plurality of proteins, comprising
- (a) performing one or more capillary electrophoretic methods, each of the one or more methods comprising
- (i) electrophoresing a sample containing multiple proteins within an electrophoretic medium contained within a capillary;
- and
- (ii) withdrawing and collecting multiple fractions containing proteins resolved during the electrophoresing step, and
- wherein each method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins;
- (b) labeling the plurality of proteins or labeling protein contained in one or more collected fractions prior to conducting the last electrophoretic method;
- and
- (c) conducting a final capillary electrophoresis method comprising electrophoresing a sample obtained from one or more fractions obtained from the preceding method in a final capillary and detecting resolved protein within, or withdrawn from, the final capillary.
5 more elements
A method for separating a plurality of proteins in an initial sample, comprising:
- (a) performing a plurality of electrophoretic methods in series, each method comprising
- electrophoresing within an electrophoretic medium a sample containing multiple proteins whereby fractions containing a subset of the multiple proteins are isolated physically, temporally or spatially, and
- wherein the sample electrophoresed is obtained from a fraction isolated during the immediately preceding method in the series, except the first method of the series in which the sample is the initial sample;
- and
- (b) detecting isolated proteins from the final electrophoretic method;
- (c) analyzing the isolated proteins to determine one or more chemical or physical characteristics of each of the isolated proteins;
- and
- (d) storing the one or more chemical or physical characteristics into a database with information about the initial sample.
5 more elements
A method for separating a plurality of proteins in an initial sample, comprising:
- (a) performing a preliminary separation of the plurality of proteins by a non-electrophoretic technique to obtain multiple fractions, wherein each fraction contains a subset of the plurality of proteins and the preliminary separation separates at least some of the plurality of proteins according to differences in amino acid sequence;
- (b) performing a plurality of electrophoretic methods in series, each method comprising
- electrophoresing a sample containing multiple proteins, whereby a plurality of resolved proteins are obtained, and
- wherein the sample electrophoresed contains only a subset of the plurality of resolved proteins from the immediately preceding method in the series, except the first electrophoretic method of the series in which the sample is from one of the multiple fractions;
- and
- (c) detecting resolved proteins from the final electrophoretic method.
3 more elements
A method for separating a plurality of proteins in an initial sample, comprising
- (a) performing a plurality of capillary electrophoretic methods in series, wherein
- the method or methods preceding the final method comprises withdrawing and collecting multiple fractions containing proteins resolved during the electrophoretic method,
- each electrophoretic method is conducted with a sample from a fraction collected in the preceding electrophoretic method, except the first electrophoretic method which is conducted with a sample containing the plurality of proteins and the final electrophoretic method which is conducted with a sample from one or more fractions obtained in the penultimate electrophoretic method;
- and
- the plurality of capillary electrophoretic methods include at least one of the methods selected from the group consisting of capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE);
- (b) labeling the proteins in the initial sample or labeling resolved protein(s) obtained from at least one of the capillary electrophoresis methods prior to conducting the last electrophoretic method, wherein
- if the plurality of methods includes a CIEF method, labeling is performed after the CIEF method;
- if the plurality of methods includes a CZE method, labeling is performed before the CZE method;
- and
- if the plurality of methods include a CGE method, labeling is performed before the CGE method;
- and
- (c) detecting labeled proteins from the final electrophoretic method.
9 more elements
A method for separating a plurality of proteins in an initial sample, comprising:
- (a) performing a plurality of capillary electrophoretic methods in series, each method comprising electrophoresing a sample obtained from the preceding method in the series, except the first method of the series in which the sample is the initial sample, whereby one or more resolved proteins are obtained, and wherein
- at least one of the methods is a capillary isoelectric focusing method in which electrophoresing is performed in an electrophoretic medium and comprises eluting resolved proteins by electroelution, electroelution comprising (i) altering the salt concentration of the electrophoretic medium, and/or (ii) pH mobilization of resolved proteins;
- and
- (b) detecting resolved protein from the final electrophoretic method.
1 more element
Dependencies are inferred from the claim text.