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SDS-polyacrylamide gel electrophoresis analysis of chitosan oligosaccharide_2745

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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.

     1.4 Imaging

     After electrophoresis, the gel will be visible under ultraviolet light imaging. Reposted elsewhere in the paper for free download http://

     2 Results and electrophoresis analysis

     2.1 The results (Figure 1,2,3,4)

     2.2 Electrophoresis diagram analysis

     2.2.1 with the literature [5] comparison we can see that this experiment is mainly the degree of polymerization of 3 to 7 of chitosan oligosaccharide with the literature [6] using thin-layer chromatography

    and liquid chromatography analysis of out of chitooligosaccharides the same.

     2.2.2 Comparison Chart 1,3, with the gum concentration increased, oligosaccharides with more and more detailed, and the more bright.

    Oligosaccharides with the position adopted by the standard oligosaccharide and glucosamine hydrochloride in contrast to be able to know the different degree of polymerization of oligosaccharides; by oligosaccharides with a bright contrast observation to know the extent of the same batch reaction system under conditions in which the shell oligonucleotide sugar up, what kinds of degree of polymerization of oligosaccharides is more dominant. From the figure we can see, in the separating gel concentration of 17.5%

    and the concentration of 3% concentrated gel, in the enzyme with the chitosan mass ratio of 4:100, the chitosan oligosaccharide obtained the largest number of degree of polymerization of 3 and 7 of the shell oligonucleotide sugar.

     2.2.3 Comparison Chart 3,4 we can see, using a continuous-

    polyacrylamide gel electrophoresis SDS get oligosaccharides with a low

    brightness and dispersion, it is difficult to a different degree of polymerization of oligosaccharides separated from the shell, while the use

    of discontinuous SDS polyacrylamide gel electrophoresis of the

    oligosaccharides to be bright with a fine and it is easy to separate the different degree of polymerization of the chitosan oligosaccharide.

     2.3 Summary

     Gum concentration in the concentration of 3%, separating gel concentration was 17.5%, the sample volume of 10 μl, a constant current

    of 45 mA, electrophoresis time of 50min under the conditions of SDS

    polyacrylamide gel electrophoresis, can achieve the most

    chitooligosaccharides excellent analysis Atlas.

     3 Discussion

     3.1 The marking process of the sample liquid temperature and time is causing a major factor in glycosidic bond breaking, time is too long or the temperature is too high will cause the molecules to be degraded

    oligosaccharides generated a lot of glucosamine or small molecular weight oligosaccharides, but the author's electrophoresis can be seen on the glucosamine and small molecular weight oligosaccharides small, indicating better conditions for the sample liquid mark.

     3.2 The composition of experimental samples buffer solution is Tris

     HCl buffer solution, 5% SDS, glycerol, bromophenol blue, compared to discontinuous electrophoresis sample buffer solution [7] less mercaptoethanol and reduces the amount of SDS, This is because ethanol is the role of thiol molecules in the disulfide bonds to break, while the disulfide bonds of sugar molecules do not exist, so do not add mercaptoethanol. SDS's role is to make sugar, bring a large number of

    negative charge, so that the size of sugar to directly determine the molecular weight of sugar in an electric field in the mobility rate. But

    the SDS itself is a kind of salt, and we obtained the oligosaccharides with a lot of salt on its own, if the SDS concentration is too high can

    lead to precipitation of sugar from the solution, thus affecting the results of observation and analysis.

     Gel concentration of 3.3 mainly by the size of sugar to determine the molecular weight, the oligosaccharides obtained in this experiment the

    sample can be used 10% to 20% of the gel. With 12.5% and 15% of the separating gel and 3% of the concentrated gel for SDS discontinuous

    polyacrylamide gel electrophoresis, the results shown in Figure 1,2 show a different degree of polymerization of the oligosaccharide and glucosamine hydrochloride bands However, relatively thick strip is not clear, the resolution is not high. Improvement for the introduction of 17.5% separating gel and 3% of the concentrated gel electrophoresis, we can

    clearly see that a single thin band, and high resolution.

     3.4 the number on the sample volume will affect the depth of bands. Along with the sample volume increases, the brightness of bands will be increasingly high, but too much will lead to the sample volume of each

    band is very bright but can not see how the major degradation product and would lead to serious tailing phenomenon, the previous sample volume is too small and that the observed dark bands would not sample solution

    concentration gradient. Screened through multiple experiments on a sample volume of 10μl, in the electrophoretic diagram can be observed a clear gradient, and there is a single clear band.

     References

     [1] Nam MY Shon YH, Kim CH, et al. Inhibitory Effect of Chitosan

    Oligosaccharides on the Growth of Tumor Cells [J]. Chit Chitos, 1999,4:184

     188.

     [2] Du Xian-guang, Zhang Mingjun, Zhang Hu, et al. Chitooligosaccharides marine engineering preparation of drug separation process and its anti-

    cancer activity of a new study [J]. Chinese Journal of Micro-Ecology,

    2001,13 (1): 5 7 .

     [3] Wen-Shui Xia, Zhang Fan, He Xin Yi. Chitooligosaccharides antibacterial activity and the role of food preservation [J]. Wuxi Light Industry Science Journal, 1998,17 (4): 10 14.

     [4] Su Chang, Wen-Shui Xia, Yao source. Papain Chitosan [J]. Wuxi Light Industry University, 2002,21 (2): 112 115.

     [5] Michael B Howard, Nathan A Ekborg, et al. Detection and Characterization of Chitinases and Other Chitin modifying Enzymes [J].

    J Ind Microbiol Biotechnol, 2003, 30: 627 635.

     [6] house, LIU Wan-shun, bit Xiao-Juan, et al. Chitosan oligosaccharide

    liquid chromatography and thin layer chromatography analysis [J]. Journal of Ocean University of China, 2005,35 (1): 113 115.

     [7] Guo Yaojun. Protein electrophoresis experimental techniques [M]. Beijing: Science Press, 1999:137. Reposted elsewhere in the paper for free download http://

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