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A kind of fluorescent protein dye and its preparation method and application

A protein and dye technology, applied in the field of chemical and biological sensing, can solve the problems of difficult decolorization of silver-stained glue, affect the clarity of the results, and consume a long time, and achieve bright color, excellent washing fastness, and high sensitivity. Effect

Active Publication Date: 2019-04-12
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When SDS-PAGE is used to analyze protein properties, Coomassie brilliant blue and silver staining are the most widely used protein stains, but they all require a separate staining and decolorization process, with cumbersome steps and high operation requirements, and silver staining is slightly Inadvertently, it will cause a deep background, which will affect the clarity of the results. The most serious thing is that some silver-stained gels are difficult to decolorize; Coomassie Brilliant Blue staining has a low staining background, but it has low sensitivity and takes a long time

Method used

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  • A kind of fluorescent protein dye and its preparation method and application
  • A kind of fluorescent protein dye and its preparation method and application
  • A kind of fluorescent protein dye and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0021] 1. The concentrated sulfuric acid that 35mL mass fraction is 98% is joined in the dry round-bottomed flask, then in the round-bottomed flask, add 3.3mL (31.85mmol) dry cyclohexanone dropwise, and it is cooled to 0 ℃, Then, in the case of vigorous stirring, add 4-diethylamino ketoacid with a total mass of 5.0136g (16mmol) in batches to the round bottom flask. Heating and reacting in medium temperature for 1.5 hours, after the reaction, the color of the reaction solution was black and red, the reaction solution was cooled to room temperature and then poured into 150g of ice, then 3.5mL of perchloric acid aqueous solution with a mass fraction of 70% was added, filtered and washed with 100mL of ice After washing with water, the obtained solid was dried in a vacuum oven at 45°C. MS (ESI+) m / z of the resulting dry product: found value 376.1909 [M+H] + , theoretical value [C 24 h 26 NO 3 ] + : 376.1907.

[0022] 2. Add 1.5mL dry N,N-dimethylformamide (DMF) into a round b...

Embodiment 2

[0026] The purposes of the fluorescent protein staining agent prepared in embodiment 1 in bovine serum albumin staining, concrete method is as follows:

[0027] Sample pretreatment: Dissolve the fluorescent protein stain completely in absolute ethanol, then add distilled water to prepare a 1mg / mL fluorescent protein stain solution; mix 50μL 1mg / mL fluorescent protein stain solution with 20μL 1mg / mL bovine Serum albumin aqueous solution was added to 25 μL of Tris-HCl buffer solution with pH 8.0, and 10 μL of glycerol was added, after thorough mixing, heated at 57° C. for 1 hour, and centrifuged at 3000 rpm for 10 min.

[0028] Electrophoresis separation: take 15 μL centrifuged supernatant and add it to the swimming lane of polyacrylamide gel (separating gel 7.5%, stacking gel 4%), add 1000mL electrophoresis buffer (3.0g Tris, 14.4g glycine, remaining distilled water), apply a constant voltage of 100V to the stacking gel part, when the bright red band runs to the separation gel ...

Embodiment 3

[0030] The purposes of the fluorescent protein stain prepared in embodiment 1 in lysozyme staining, specific method is as follows:

[0031] Sample pretreatment: Dissolve the fluorescent protein stain completely in absolute ethanol, then add distilled water to prepare a 1mg / mL fluorescent protein stain solution; dissolve 50μL 1mg / mL fluorescent protein stain solution and 20μL 1mg / mL The enzyme aqueous solution was added to 25 μL of Tris-HCl buffer solution with pH 8.0, and 10 μL of glycerol was added. After mixing well, it was heated at 57°C for 1 hour to fully denature the lysozyme, and centrifuged at 3000 rpm for 10 min.

[0032] Electrophoresis separation: take 15 μL of centrifuged supernatant and add it to the swimming lane of polyacrylamide gel (16.5% separating gel, 10% interlayer gel, 4% stacking gel), and add 1000mL electrophoresis buffer (3.0g Tris, 14.4g glycine, the remainder is distilled water), electrophoresis was carried out at a temperature of 4°C and a current o...

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Abstract

The invention discloses a fluorescent protein dyeing agent as well as a preparation method and application of the fluorescent protein dyeing agent. The structural formula of the fluorescent protein dyeing agent is as shown in the specification. A solution of the fluorescent protein dyeing agent has intense fluorescence, the dyeing agent comprises aldehyde groups which can be reacted with amino on protein to form covalent bonds for dyeing the protein, so that the dyeing agent has the advantages of being firm in dyeing, not liable to decolor, convenient to use, few in operation steps, short in time and high in sensitivity, is capable of improving the dyeing and fluorescence detection effect, and is beneficial to fluorescence or naked-eye recognition and detection.

Description

technical field [0001] The invention belongs to the technical field of chemical and biological sensing, and in particular relates to a protein stain with fluorescent properties, as well as a preparation method and application of the protein stain. Background technique [0002] Organic small molecule fluorescent probes are widely used in the fields of environmental science, life science and material science, and have increasingly become an indispensable research method in the fields of modern life science and disease diagnosis. Therefore, the development of functional fluorescent dye molecules with practical value much attention. Molecular fluorescent probes based on organic fluorophores have many advantages such as high sensitivity, easy operation, good reproducibility, good membrane permeability, and in situ detection. Moreover, fluorescent dyes are often used as fluorescent probes, and have important applications in the fields of bioengineering and medical detection for l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D311/82C09K11/06G01N1/30C12Q1/34
CPCC07D311/82C09K11/06C09K2211/1007C09K2211/1088C12Q1/34C12Q2334/40G01N1/30G01N2001/302
Inventor 吕家根段瑞赵春欣张胜海闫思谕
Owner SHAANXI NORMAL UNIV
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