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A method of electrophoresis

An electrophoretic separation and charge technology, applied in the field of separation of protein and/or peptide components by electrophoresis, can solve problems such as unsatisfactory solutions to this problem

Inactive Publication Date: 2005-08-17
GE HEALTHCARE BIO SCI CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As already discussed, this type of alkylation is not ideal for solving the problem

Method used

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  • A method of electrophoresis

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Experimental program
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Effect test

Embodiment 1

[0129] Clean Gel TM 25 (Amersham Biosciences, Uppsala, Sweden), a commercially available dry gel for zone electrophoresis, with a size of 250×110×0.5 mm (after rehydration), prepared by T=5% (5 g Monomer / 100ml) and C=3% (3 g crosslinker / 100 g monomer) stacking gel and T=10 and C=2 separating gel, which were cut in half. One half was rehydrated in a rehydration solution delivered by gel containing 0.3M Tri-acetic acid, pH 6.5 buffer and 0.1% SDS. The other half was rehydrated in the corresponding solution to which bis(hydroxyethyl)disulfide was also added at a concentration of 300 mM. Place the two gel halves in a MultipHor at 15°C TM on a cooling plate (Amersham Biosciences, Uppsala, Sweden). The cathode paper core contains Tris-ribinoic acid-SDS, and the anode paper core contains Tris-acetic acid (electrode solution delivered by gel). Dissolve 0.3 mg / ml of bovine serum albumin, chicken ovalbumin, soybean trypsin inhibitor and bovine carbonic anhydrase in 0.375M Tris / HCl,...

Embodiment 2

[0133] Immobiline DryStrip, pH 6-11, 18 cm long TM , rehydrated overnight with a solution containing 8M urea, 0.5% CHAPS, 1% IPGTM buffer, pH 6-11, and the redox chemicals defined below. Samples extracted from mouse liver containing 50 mM Tris, 7 M urea, 2 M thiourea, 4% (w / v) CHAPS, and 10 mM DTT were diluted with the same solution used for rehydration (10 μl diluted to 160 μl) to provide the final concentration A sample of 1 mg / ml. 80 μl of each sample, corresponding to the rehydrated IPG-strip, was applied to the sample cup adjacent to the anode end of the strip. Silver staining of gels generated in the second dimension is shown in figure 2 a-f.

[0134] Fig. Sample and rehydration solution

[0135] a 8M urea, 0.5% CHAPS, 1% IPG buffer pH6-11, 20mM mercaptoethanol

[0136] b 8M urea, 0.5% CHAPS, 1% IPG buffer pH6-11, 20mM dithiothreitol

[0137] c 8M urea, 0.5% CHAPS, 1% IPG buffer pH6-11, 50mM bis-(2-hydroxyethyl)-disulfide

[0138] d 8M urea, 0.5% CHAPS, 1% IPG buf...

Embodiment 3

[0143] Immobiline DryStrip pH 7.5-9.5, 24 cm long TM , rehydrated overnight with a solution containing 8M urea, 0.5% CHAPS, 1% IPGTM buffered pH 6-11 and redox chemicals as described below. Samples containing murine liver proteins were diluted with the same solution used for rehydration (10 μl diluted to 160 μl) to obtain samples with a final concentration of 1 mg / ml. 80 μl of each sample, corresponding to the rehydrated IPG-strip, was applied to the sample cup adjacent to the anode end of the strip. The gels produced in the second dimension were silver-stained and the results are shown in panels h–j.

[0144] Fig. Sample and rehydration solution

[0145] H 8M urea, 0.5% CHAPS, 1% IPG buffer pH8-10.5, 20mM mercaptoethanol

[0146] I 8M urea, 0.5% CHAPS, 1% IPG buffer pH8-10.5, 50mM bis-(2-hydroxyethyl)-disulfide

[0147] J 8M urea, 0.5% CHAPS, 1% IPG buffer pH8-10.5, 50mM bis-(3-hydroxypropyl)-disulfide

[0148] This experiment shows that the addition of disulfides accord...

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Abstract

The present invention relates a method of electrophoretic separation of protein and / or peptide components of a sample in a convection stabilised medium. More specifically, the method comprises the steps to contact the sample with the separation medium; to apply a voltage across said medium; and to observe the results by analysis of one or more sections of the separation medium. In the present method, a disulphide-comprising compound is added before or during the procedure to make an excess of reactive disulphide groups accessible to react with the cysteine groups of the proteins and / or peptides all through the separation procedure. The present invention also relates to electrophoretic separation media that comprises reactive disulphide groups, such as polyacrylamide gels, and the use of a solution that comprises reactive disulphide groups to pretreat an electrophoretic separation medium.

Description

technical field [0001] The invention relates to the field of electrophoresis, in particular to a method for separating protein and / or peptide components by electrophoresis. The present invention also relates to a method for pretreatment of the separation medium used in the above method of the present invention, and a kit for the pretreatment. Background technique [0002] The separation of biomolecules, such as proteins and peptides, has attracted increasing interest over the past many years. As the final step in bioprocessing methods for the preparation of these substances, for example for the preparation of protein-based pharmaceutical compounds, some biomolecules need to be isolated. Likewise, for analytical purposes, biomolecules need to be separated so that the proteins and / or peptides in the sample can be quantified and characterized, and electrophoresis is often used for the separation step. A number of methods can be used to measure and quantify isolated proteins. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D57/02B03C5/00C07K1/26G01N27/447
CPCG01N27/44747B01D57/02C07K1/26
Inventor B·比耶尔奎斯特I·奥尔松R·帕尔姆格伦
Owner GE HEALTHCARE BIO SCI CORP
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