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SDS-polyacrylamide gel electrophoresis analysis of chitosan oligosaccharide_2745

By Gloria Black,2014-10-31 18:43
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SDS-polyacrylamide gel electrophoresis analysis of chitosan oligosaccharide_2745

    SDS-polyacrylamide gel electrophoresis analysis of chitosan oligosaccharide

     Authors: Wang Liang, Wuchang Ying, Hua Liu, Zhang Tao

     Abstract Objective To study the shell of a fast and accurate analysis of the qualitative determination of the composition of oligosaccharides. Methods Continuous electrophoresis and discontinuous electrophoresis chitooligosaccharides comparative experiments to determine the best analytical conditions. The results concentrated gel concentration of 3%,

    separating gel concentration was 17.5%, the sample volume of 10 μl, a

    constant current of 40 mA, electrophoresis time of 50 min under the conditions of the best chitosan oligosaccharide analysis of patterns. Conclusion SDS polyacrylamide gel electrophoresis analysis of the

    composition of chitosan oligosaccharide is indeed a simple way to quickly and efficiently qualitative detection methods.

     Key words chitosan oligosaccharide; electrophoresis analysis; polyacrylamide gel electrophoresis

     Abstract Objective To explore a qualitative, fast and accurate way for analyzing composition of chitooligosaccharide. Methods By consecutive electrophoresis and non consecutive electrophoresis

    experiments, the best analysis conditions of chitooligosaccharide were

    determined. Results The concentration of plastic was 3 percent, the concentration of separation of plastic was 17.5 percent, the volume of samples was 10 μl, constant current was 40 mA, electrophoresis time was 50 min. Best analysis photos of chitooligosaccharide Can be obtained under those conditions. Conclusion The method of analyzing composition of chitooligosaccharide is truly a simple, effective, accurate and qualitative way.

     Key words chitooligosaccharide; electrophoretic analysis;

    polyacrylamide gel electrophoresis

     Shell chitosan oligosaccharide obtained by the hydrolysis product of D glucosamine by β (1 4) glycosidic bond connecting

    glucopyranosyl bond made of 2 to 10 units of oligosaccharides. Reported in

    the literature [1 3], chitosan oligosaccharide with the promotion of spleen antibody production, inhibit tumor growth and other physiological functions, in the food and daily chemical products also have a wide range of applications. Detection of more than ordinary chitosan oligosaccharide

    by thin-layer chromatography and high performance liquid chromatography. For the thin-layer chromatography, while prices are low, but the operation for a long time can not be quantified, while the high-performance liquid

    chromatography, the effect is good, but it needs large-scale equipment and

    the mobile phase, expensive for the general lab the initial screening is negative. Therefore, the need for a simple and efficient low-cost method

    of testing chitooligosaccharides. This study used SDS oligosaccharides

    polyacrylamide gel electrophoresis, fast, easy to make up for the shortcomings of traditional detection methods, with better application and popularization.

     An experimental part of the

     1.1 Instrument and reagents

     Oligosaccharides (Sigma), glucosamine hydrochloride (China Pharmaceutical Group Shanghai Reagent Company), chitosan (deacetylation degree ? 90%, Shanghai Boao Bio-Technology Co., Ltd.), snail enzyme

    (Lvshengyuan Biotechnology Company), sodium borohydride nitrile (Fluka),

    the other reagents were of analytical grade.

     Freeze-drying machine (Thermo Corporation), high-speed refrigerated

    centrifuge (Eppendorf Inc.), pH meter (Chengdu ARCA Technology Development Company), 10kD ultrafiltration tubes (Millipore Corporation), MINI

    PROTEIN ? electrophoresis system (Bio Rad Company), Gel imaging system

    (Gene Company).

     1.2 Methods

     1.2.1 Degradation of Chitosan

     According to reference [4] method, taking 1% chitosan acetic acid

    solution, add 1 mg / ml of enzyme solution at 40 ? in water bath pot

    reaction of 100 min, boiled 10 min to terminate reaction, plus 1 mol / L NaOH solution, 4 000 r / min centrifugation 5 min, then centrifuged at 10 kD ultrafiltration tube, 3 000 r / min centrifugation 10 min, collecting

    the filtrate under vacuum freeze-drying.

     1.2.2 Solution Preparation Methods marked anthranilic acid 0.28g, dissolved in 0.63g of sodium borohydride nitrile 10ml water, dissolved in water at 65 ? (the solution prepared before use).

     1.2.3 Preparation of sample solution according to reference [5] method of taking the above-mentioned experimental steps had been freeze-

    dried samples, and the oligosaccharides of glucosamine hydrochloride, 0.01g, were added to 0.2ml mark has been preheated solution, 65 ? of the

    water bath pot heat 2 h after adding 1.2 ml acetone, at 15 000 r / min centrifugation 3 min, to collect sediment and add 0.05ml sample buffer (0.08 mol / LTris HCl buffer solution, pH6.8 5% SDS 87% Gan Oil

    bromophenol blue), mixing.

     1.3 Electrophoresis

     According to reference [6] method of preparation of 12.5%, respectively, 15%, 17.5% separating gel and 3% of the concentrated gel.

     Canadian sample: Take 1.2.3 obtained 10 μl of sample solution for

    SDS polyacrylamide gel electrophoresis.