Protein and gamma-polyglutamic acid simultaneous coloring counterstaining method

A polyglutamic acid and protein technology, applied in the preparation of test samples, measuring devices, instruments, etc., to achieve the effect of convenient and quick operation

Inactive Publication Date: 2013-08-28
SHANDONG UNIV OF TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Select the electrophoresis gel concentration and electrophoresis conditions according to the characteristics of the protein and γ-polyglutamic acid samples for electrophoresis

Method used

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  • Protein and gamma-polyglutamic acid simultaneous coloring counterstaining method

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Embodiment 1

[0019] Example 1 Coomassie Brilliant Blue staining, Coomassie Brilliant Blue and Methylene Blue counterstaining, and silver staining were performed after SDS-PAGE of γ-polyglutamic acid samples and low molecular weight standard protein samples.

[0020] (1) Preparation of reagents used in this example: 30% electrophoresis gel mother solution: 29.2 g acrylamide, 0.8 g methylenebisacrylamide, distilled water to 100 ml, filtered through three layers of filter paper and stored at 4 °C in the dark.

[0021] 1.5 M pH=8.8 Tris-HCl: add 45.43 g of Tris to 200ml with double distilled water, adjust the pH to 8.8 with 6mol / L concentrated HCl, and dilute to 250ml. Store at 4°C.

[0022] 0.5 M pH6.8 Tris-HCl: Add 6.0 g Tris to 80 ml with double distilled water, adjust the pH to 6.8 with 6 mol / L HCl, and then dilute to 100 ml. Store at 4°C.

[0023] 10% SDS: 10 g SDS plus 50 ml double distilled water. 60 ℃ water bath to aid dissolution, dilute to 100ml, store at room temperature.

[002...

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Abstract

The present invention provides a protein and gamma-polyglutamic acid simultaneous coloring counterstaining method, which is an electrophoresis staining method belonging to the field of biotechnology, and can allow the protein on gel and gamma-polyglutamic acid to be chromogenic simultaneously. The method is characterized by adopting the following steps: (1) preparing protein and gamma-polyglutamic acid electrophoresis samples and performing polyacrylamide gel electrophoresis, (2) fixing the gel after electrophoresis, firstly staining with Coomassie brilliant blue R250 and bleaching, (3) placing the gel after the first staining in a methylene blue dye liquor for re-staining and bleaching until the protein and gamma-polyglutamic acid strips are clear. Compared with the prior art, the present invention solves the problem of simultaneous staining of protein and gamma-polyglutamic acid, can determine molecular weight and purity thereof according to the strip band position and the strip band number of the stained gamma-polyglutamic acid, and is convenient and fast to operate.

Description

technical field [0001] The invention provides a counterstaining method for simultaneous coloring of protein and γ-polyglutamic acid, which belongs to the field of biotechnology and is an electrophoretic gel staining method, which can simultaneously develop color of protein and γ-polyglutamic acid on the same piece of gel . Background technique [0002] γ-PGA is an extracellular anionic polyamino acid produced by microorganisms and is the main component of some bacterial capsules. In 1937, Ivnoavics first discovered γ-PGA in the capsule of Bacillus anthracsi. γ-PGA is a high molecular polymer formed by D-glutamic acid (D-Glu) and L-glutamic acid (L-Glu) through α-amino and γ-carboxyl in the form of peptide bonds. γ-PGA is usually composed of about 5,000 glutamic acid monomers, and the basic skeleton is in the form of straight chain fibers. Generally speaking, the average molecular weight (Mw) of γ-PGA produced by Bacillus fermentation is between 100-1000 kD. The structure...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N27/447
Inventor 刘文胡巍董建
Owner SHANDONG UNIV OF TECH
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