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Method and device for separation of charged molecules by solution isoelectric focusing

a technology of isoelectric focusing and charged molecules, applied in the direction of fluid pressure measurement, liquid/fluent solid measurement, peptide measurement, etc., can solve the problems of low maximum sample loading capacity, lack of resolution and dynamic range for resolving and detecting large numbers of charged molecules, and high cost of separation

Inactive Publication Date: 2005-04-28
WISTAR INSTITUTE
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  • Abstract
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Benefits of technology

[0013] This invention provides a novel small-scale solution isoelectric focusing device and method that can reproducibly fractionate charged molecules into well-defined pools. This approach can be applied to complex charged molecule samples, such as eukaryotic proteome samples where reproducible resolution and quantitation of greater than 10,000 protein components is feasible.

Problems solved by technology

Current two dimensional (2D) methods, however, lack both adequate resolution and sufficient dynamic range for resolving and detecting large numbers of charged molecules, for example, the protein components present in eukaryotic proteomes which can comprise over 10,000 proteins.
A major disadvantage of existing 2D gel methods when applied to a large number of charged molecules is that the maximum sample loading capacity is fairly low, which results in detection of only the most abundant charged molecules when currently available stains are used (Herbert et al.
Increasing the amount of sample above an optimal level results in horizontal streaking of many proteins.
Although current IPG-based 2D gels have much higher resolution than alternative separation methods, not all charged molecules in a sample can be resolved by a single IPG gel.
This incomplete resolution contributes to errors in subsequent quantitation and identification of charged molecules.
The use of these methods, however, can result in an incomplete separation of charged molecules between fractions and a poor yield.
Using current methods cross contamination of specific charged molecules between fractionated pools can seriously complicate quantitative analyses and comparisons, since many charged molecules appear in more than one fraction and the degree of cross contamination is often highly variable.
However, this apparatus has no separation barriers and is typically a low resolution technique with relatively large volumes for individual fractions.
While the IsoPrime™ unit can provide high quality separations, its large volume and design make it impractical for prefractionation of samples containing complex mixtures of charged molecules, especially under denaturing conditions.
Similarly, other preparative isoelectric focusing instruments suffer from at least several of the limitations encountered with either the Rotofor™ or the IsoPrime™; specifically these instruments: (1) require a large sample volume, (2) produce large volume, dilute fractions that need to be concentrated with attendant losses, (3) exhibit poor resolution, or (4) involve expensive, complex instrumentation.

Method used

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  • Method and device for separation of charged molecules by solution isoelectric focusing
  • Method and device for separation of charged molecules by solution isoelectric focusing
  • Method and device for separation of charged molecules by solution isoelectric focusing

Examples

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example 1

Preparation of Metabolically Radiolabeled Escherichia coli Extracts

[0054] Metabolically radiolabeled E. coli extracts were used in these studies to systematically evaluate protein recoveries. E. coli was selected since this relatively simple organism can be readily metabolically radiolabeled to high specific activity to provide sensitive and reliable detection of protein losses. In contrast to chemical labeling methods such as iodination of a portion of the sample, metabolic radiolabeling of the entire sample ensured that the labeling method would not alter the properties of the proteins and that each protein was a homogeneous population of molecules.

[0055]E. coli were cultured as previously described (Harper & Speicher, (1995) in CURRENT PROTOCOLS IN PROTEIN SCIENCE (Coligan et al., eds.), pp. 6.6.1-6.6.21, John Wiley & Sons Inc., New York) with modifications. Briefly, E. coli cells were inoculated in Luria broth (LB medium) and incubated at 37° C. with continuous shaking (250-30...

example 2

Two-Dimensional Electrophoresis

[0057] Isoelectric focusing equipment, IPG gels, and relevant reagents were purchased from Amersham Pharmacia Biotech (San Francisco, Calif., USA), unless otherwise indicated. Proteins were isofocused using different pH range IPG strips (pH 3-10 NL, 4-7L and 4.8-6.2L, 18 cm length) on the IPGphor™ Isoelectric Focusing System. Narrow pH range IPG gels (pH 4.8-6.2L) were cast in the laboratory using cornmercial immobilines as detailed in the IPG application manual. Immediately prior to isoelectric focusing, dried IPG strips were rehydrated for 8 h with sample in IPG sample buffer (350 μl) in the ceramic strip holders (1 h without current followed by 7 h at 30 Volts) as described by Gorg et al. (1999) Electrophoresis 20, 712-717. The IPG sample buffer contained 2 M thiourea, 7 M urea, 0.1 M DTT, 4% CHAPS and 2% IPG-buffer (carrier ampholyte mixture matching the pH range used). After the 8 h rehydration, samples were focused for 1 h each at 500 V, 1000 V,...

example 3

Determination of Protein Recoveries

[0059] Protein recoveries and losses throughout alternative prefractionation methods were determined using liquid scintillation counting. Any surfaces that contacted samples were extracted with 1% SDS to remove any adsorbed or precipitated proteins. Typically, a small volume of these SDS extracts or sample solutions (5 μl) was mixed with 4.5 ml of Bio-Safe II scintillation cocktail (Research Products International Corp, Mt. Prospect, Ill., USA) and radioactivity was counted using a Model 1500 TRI-CARB Liquid Scintillation Analyzer (PACKARD Instrument Company, Downers Grove, Ill., USA). The radioactivity left in gels after elution was counted after solubilization using 1 N NaOH at 60° C. for 3 h, followed by neutralization with concentrated HCl and addition of the scintillation cocktail.

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Abstract

The invention provides a novel solution isoelectric focusing device and method that can reproducibly fractionate charged molecules into well-defined pools. This approach can be applied to mixtures of charged molecules, such as eukaryotic proteome samples where reproducible resolution and quantitation of greater than 10,000 protein components is feasible.

Description

STATEMENT OF GOVERNMENT INTEREST [0001] This invention was made with government support under NIH Grant No. RO1 CA77048 and RO1 CA66671. As such, the government has certain rights in this invention.TECHNICAL FIELD OF THE INVENTION [0002] The invention relates to the field of separation of charged molecules, and in particular the separation of mixtures of charged molecules. BACKGROUND OF THE INVENTION [0003] Complex charged molecule mixtures, such as protein mixtures can be separated by isoelectric focusing under denaturing conditions in gel tubes or strips that contain either soluble ampholytes (Klose, (1975) Humangenetik 26, 231-243; O'Farrell, (1975) J. Biol. Chem. 250, 4007-4021; Scheele, (1975) J. Biol. Chem. 250, 5375-5385) or immobilines (Bjellqvist et al. (1982) J. Biochem. Biophys. Meth. 6, 317-339). For quantitative comparisons in changes in total protein profiles a second dimension separation can be done on a conventional SDS polyacrylamide gel electrophoresis (PAGE) slab ...

Claims

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Application Information

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IPC IPC(8): C07K1/28C08F2/58G01N27/447
CPCG01N27/44747G01N27/44795G01N27/44773
Inventor SPEICHER, DAVID W.ZUO, XUN
Owner WISTAR INSTITUTE
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