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Method and device for separation and depletion of certain proteins and particles using electrophoresis

A separation area, flow electrophoresis technology, applied in the direction of measuring devices, material analysis by electromagnetic means, analysis of materials, etc., can solve difficult electrophoretic separation/removal of analytes to maintain sufficient stability, no given, no instructions can be removed repeatedly High-abundance proteins and other issues

Active Publication Date: 2009-08-19
BECTON DICKINSON & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to the best of the inventors' knowledge, no successful and reproducible selective removal process using this technique has been disclosed in the art, not least because of the lack of the same conditions that must be provided in the separation chamber of an FFE device. Information about, and difficulty in maintaining sufficiently stable conditions during electrophoretic separation / removal of analytes from samples
[0025] In this context, it should be noted that although US applications US 2004 / 050697 and US 2004 / 050698 to Eckerskorn et al. Achieving successful and reproducible removal of a given high-abundance protein (e.g., albumin) also does not give information on the selection of an appropriate separation buffer system, the pH conditions or profiles employed, or the maintenance of stable conditions in FFE equipment. Any teaching on the stable medium

Method used

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  • Method and device for separation and depletion of certain proteins and particles using electrophoresis
  • Method and device for separation and depletion of certain proteins and particles using electrophoresis
  • Method and device for separation and depletion of certain proteins and particles using electrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0261] Example 1: Separation of human plasma according to the DFE protocol

[0262] The examples illustrate the separation of high abundance proteins (human serum albumin, HSA, HSA) from human plasma samples using the DFE protocol using a gel-free, support-matrix-free or carrier-free FFE electrophoresis method and equipment suitable for carrying out the method. ). The non-denatured human plasma was diluted 1:10 with the medium of the medium inlet 4 of the device as shown in FIG. 1A and injected or introduced into the separation zone via the sample inlet 4 at a sample load rate of 5 ml / h.

[0263] Introduce the following media to the device:

[0264] Media inlet 1 and 2: 100mM sulfuric acid + 10% glycerol (pH1.30)

[0265] Media inlet 3: 200mM 2-amino-butyric acid, 100mM gluconic acid, 50mM pyridineethanesulfonic acid (PESS), 30mM glycylglycine, 10% glycerol (pH3.39)

[0266] Media inlet 4: 30mM MES, 100mM glycylglycine, and 10% glycerol (pH4.92)

[0267] Media inlet 5: 200...

Embodiment 2

[0276] Example 2: Separation of human plasma according to the DSE protocol

[0277] This example illustrates the separation of high-abundance proteins (human serum albumin, human serum albumin, HSA). The non-denatured human plasma was diluted 1:10 with the medium of the medium inlet 4 of the apparatus shown in Figure 4, and injected or introduced into the separation area through the sample inlet 4 at a sample loading rate of 5 ml / h.

[0278] Introduce the following media to the device:

[0279] Media inlet 1: 100 mM sulfuric acid; 10% glycerol

[0280] Media inlet 2: 100 mM sulfuric acid; 10% glycerol

[0281] Media inlet 3: 25% BD FFE Separation Buffer 1 + 10% Glycerol

[0282] Media inlet 4: 30 mM MES; 100 mM glycylglycine (glygly); 14% BDFFE separation buffer 2; 10% glycerol

[0283] Media inlet 5: 25% BD FFE Separation Buffer 2+10% Glycerol, (pH6.94)

[0284] Media inlet 6: 150mM NaOH+50mM ethanolamine, 10% glycerol

[0285] Medium inlet 7: 150mM NaOH+50mM ethanolam...

Embodiment 3

[0292] Example 3: Parallel DFE separation of analytes from two samples

[0293]This example illustrates the simultaneous separation of high-abundance proteins (human serum albumin, HSA, and ). The scheme adopted in this embodiment, starting from the anode to the cathode, employs the following media: anode stabilization media, a first separation zone containing a first pH function and a first pH separation plateau, an interelectrode stabilization media (which also acts as an adjacent separation zone 1 The focusing medium of the pH separation plateau of and 2), the second separation zone containing the pH separation plateau and the second pH function, and the cathode stabilization medium.

[0294] Dilute the first non-denatured human plasma sample 1:10 with the medium of medium inlet 2, and inject or introduce it into the separation area through the sample inlet located near the medium inlet 2 at a sample loading rate of 5 ml / h, while using the medium inlet 6 The medium dilute...

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Abstract

The present invention provides a novel and advantageous method for separating analytes by free flow electrophoresis. The methods are particularly suitable for depleting major constituents such as albumin from samples of biological origin, optionally combined with a further separation of the remaining portion of the sample. The sample portions recovered from the method can be used advantageously in downstream applications such as 1D or 2D-PAGE, HPLC or mass spectrometric analysis. Also provided are buffer systems, kits comprising such buffer systems, and devices for carrying out the free flow electrophoretic separation methods of the present invention.

Description

technical field [0001] The present invention relates to methods and devices for continuous, support-free offset electrophoresis involving separation conditions and media that enable the separation and possible removal of certain analytes with different pIs. Background technique [0002] Electrophoresis is a well-established technique for separating particles based on the migration of charged particles under the action of a direct current. As variations on the above principles, several different modes of operation have been developed, such as isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP), which are well known to those skilled in the art of. [0003] Among electrophoretic techniques, free-flow electrophoresis (FFE) is one of the most promising techniques [Krivanova L. & Bocek P. (1998), "Continuous free-flow electrophoresis", Electrophoresis 19: 1064-1074]. FFE is a technique in which the separation of analytes occurs in a carrier-free medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
CPCG01N27/44795G01N27/44769
Inventor 格哈德·韦伯
Owner BECTON DICKINSON & CO
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