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Application of berberine and its derivates in protein fluorescent detecting

A protein fluorescence, berberine technology, applied in the field of protein fluorescence detection, can solve the problem of no protein gel fluorescence staining, and achieve the effect of ensuring high selectivity and sensitivity, low cost, and improving safety

Inactive Publication Date: 2008-01-30
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there are no reports of its use for fluorescent staining on protein gels after electrophoresis

Method used

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  • Application of berberine and its derivates in protein fluorescent detecting
  • Application of berberine and its derivates in protein fluorescent detecting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The dyeing effect comparison of embodiment 1 different dyestuffs

[0028] The protein sample in this example is SDS-6H standard protein (Sigma).

[0029] Berberine protein fluorescent staining method:

[0030] 1) Place the protein sample gel after SDS-PAGE electrophoresis in a fixative for 5 minutes, discard the fixative, the fixative is 40% ethanol and 10% aqueous acetic acid by volume, and rinse with deionized water 3min;

[0031] 2) Add fluorescent staining solution for staining for 5 minutes, rinse with deionized water for 3 minutes, the staining solution is an aqueous solution containing 0.0005% (w / v) berberine dye and 2% acetic acid, and then rinse with deionized water for 0.5 minutes;

[0032] 3) Detection. Place the stained gel on a transilluminator, transmit at 312nm, and record the image.

[0033] Carry out protein staining with hydrochloride, sulfate, bisulfate, nitrate, tannate, hydroiodide of berberine respectively according to the above-mentioned metho...

Embodiment 2

[0044] Example 2 E.coli BL21 total protein two-dimensional electrophoresis staining

[0045] Take the E.coli BL21 bacterial liquid, collect the bacterial cells by centrifugation at 12000 rpm, and add 0.02mol / L phosphate buffer (pH7.0) to resuspend the bacterial cells. Cells were disrupted using a sonicator. Centrifuge at 18,000 rpm for 30 minutes, collect the supernatant, and obtain the total protein of E.coli. After the above total protein is subjected to two-dimensional gel electrophoresis, it is stained with berberine according to the method of Example 1, and the result is shown in FIG. 2 . It can be seen from Figure 2 that this staining method is basically consistent with the visualized protein spots of SYPRO Red staining method, and this method is faster and more sensitive.

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Abstract

The invention relates to a protein fluorescence detection technology, in particular to the application of the berberine and the derivatives in the protein fluorescence detection. The invention also provides a method to apply the berberine to the protein fluorescence gel detection, and the method comprises the steps of: fixing the electrophoresis gel of the protein sample in the stationary liquid, washing by the deionized water; dyeing, washing by the deionized water; detection. The invention has the advantages of high sensitive, simple and rapid operation, good reproducibility, good dyeing background, good linear relationship, good reversibility, good compatibility, safe using and low cost, etc.

Description

technical field [0001] The invention relates to protein fluorescence detection technology, in particular to a new fluorescent dye of protein. Background technique [0002] In the field of life science research, it is inseparable from the research on protein and DNA, the basic substances of life, and also inseparable from the detection technology of protein. Especially in recent years, with the rapid development of genomics and proteomics, we have higher requirements for protein separation and detection technology. [0003] There are many protein detection methods, including turbidimetry, Kjeldahl method, absorbance photometry, fluorescence photometry, and the newly developed resonance Rayleigh scattering method, high performance liquid chromatography, and capillary electrophoresis analysis. Fluorescence photometry is currently the most researched, most widely used, easy-to-operate, and highly sensitive detection method. [0004] Berberine is an isoquinoline alkaloid with t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/52G01N21/76C09K11/06
Inventor 金利泰
Owner WENZHOU MEDICAL UNIV
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