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The use of single blue a as a protein prestain

A pre-staining agent and protein technology, which is applied in the field of single blue A as a protein pre-staining agent, can solve the problems of high cost and many detection steps, and achieve the effect of moderate sensitivity, simple operation and low price

Active Publication Date: 2015-11-25
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of Western-blot is to use gel electrophoresis to separate native or denatured proteins, then transfer the proteins to the membrane, use unlabeled specific primary antibodies to bind to the antigens transferred to the membrane, and then add labels (radiogen, Biotin-labeled) secondary antibodies for hybridization detection, there are many detection steps, high cost and other defects, which need to be improved

Method used

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  • The use of single blue a as a protein prestain
  • The use of single blue a as a protein prestain
  • The use of single blue a as a protein prestain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1 Various staining methods and protein purification after staining with single blue A staining protein

[0097] 1. Method of staining protein with single blue A

[0098] 1.1 Heating catalyzed dyeing reaction

[0099] (1) Mix 10 μl single blue A staining solution with 90 μl protein solution.

[0100] (2) Heat the mixed sample at 100°C for 1 min.

[0101] (3) Then add 100 μl of dyeing optimizer and heat at 100°C for 1 min.

[0102] (4) After the sample is cooled to room temperature, add 20 μl staining terminator and let stand at room temperature for 5 minutes.

[0103] 1.2 Electrical stimulation catalyzed staining reaction

[0104] (1) Take 10 μl single blue A staining solution and 90 μl 10% BSA protein solution and mix thoroughly.

[0105] (2) Add the mixed sample to a 200ml electro-cup.

[0106] (3) The electrorotator selects 1800V as the output voltage and shocks for 20ms.

[0107] (4) Then add 100 μl dyeing optimizer and let stand at room temperature fo...

experiment example 1

[0128] Experimental example 1 Preparation and identification of prestained protein markers with single blue A dye

[0129] 1 Experimental method

[0130] 1.1 marker protein mix

[0131] Add 0.1 μg of Taq enzyme protein, BSA protein, lactate dehydrogenase A protein and IL-7 into 1.5ml EP tubes and mix thoroughly to obtain unstained mixed protein molecular weight standard protein (marker protein).

[0132] 1.2 Marker protein staining

[0133] (1) Add 3 μl of Mono Blue A dye and mix thoroughly.

[0134] (2) Heat the mixed sample at 100°C for 1 min.

[0135] (3) Then add 30 μl of dyeing optimizer and heat at 100°C for 1 min.

[0136] (4) After the sample is cooled to room temperature, add 0.6 μl staining terminator and let stand at room temperature for 5 minutes.

[0137] 1.3marker protein purification

[0138] (1) Weigh 5g of Dextran G-50, dilute to 50ml with deionized water, boil in boiling water for 3h.

[0139] (2) Put the well-stirred gel into a 0.5ml EP tube with a ho...

experiment example 2

[0153] Experimental example 2 Gradient detection experiment of single blue A labeled bovine serum albumin

[0154] 1. Experimental method

[0155] Take three 1.5ml EP tubes respectively, add 10 μl, 1 μl, 0.1 μl of BSA protein with a concentration of 1 μg / μl, and then add 9 μl of double-distilled water to the EP tube with 1 μl of BSA protein at a concentration of 1 μg / μl, and add 0.1 Add 9.9 μl of double-distilled water to the EP tube of BSA protein with a concentration of 1 μg / μl, and mix thoroughly. Take 1 μl of the solution from each tube, and add 1 μl of monoblue A dye (200mM) respectively, and heat at 100°C for 1min. After the samples were cooled, 10 μl of dyeing optimizer was added to each tube, and heated at 100°C for 1 min. After the 3 tubes were cooled to room temperature, 2 μl of staining terminator was added respectively, and the tubes were allowed to stand at room temperature for 5 minutes. Subsequent identification by SDS-PAGE electrophoresis.

[0156] 2. Exper...

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Abstract

The invention discloses an application of single blue A as a protein pre-staining agent, belonging to the field of protein staining. According to the application disclosed by the invention, adopting single blue A as a protein pre-staining agent has the advantages of simple and convenient staining steps, short staining time, small staining agent dosage and the like; and the stained protein product can be directly detected by eyes and also can be detected by a scanning instrument under near-infrared exciting light. When the single blue A is used for staining protein, the staining reaction can be promoted and the staining efficiency can be improved by adopting base catalysis or electric shock catalysis; and the technological parameters of base catalysis or electric shock catalysis are further optimized. The application disclosed by the invention has broad application prospects in preparation of protein molecular weight standard products, optimization of the detection way of Western-blot, In-Gel Western or tube polyacrylamide gel electrophoresis, and observation of the positioning or distribution condition of target protein in cells.

Description

technical field [0001] The present invention relates to the new use of single blue A, in particular to the new use of single blue A as a protein prestaining agent, and belongs to the application field of single blue A. Background technique [0002] Proteomics refers to taking all the proteins encoded by the genome as the research object, studying the composition and changing rules of proteins from the cellular level and the overall level, so as to deeply understand the various physiology and pathology of the organism. Compared with traditional protein research, proteomics research embodies the characteristics of comprehensiveness, integrity, high throughput and large scale. The study of proteomics plays an irreplaceable role in the empirical analysis of the proteome that has been theoretically predicted by the genome project. More importantly, proteome analysis does not need to rely on genome research. Using protein research is expected to find sequences from protein express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/30
Inventor 丁卫窦云鹏龚嘉玲张晨光郑少鹏油红捷
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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