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
1.2.1 Degradation of Chitosan
According to reference  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  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.
According to reference  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.