• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Derivatization of Free Natural Glycans for Incorporation onto Glycan Arrays: Derivatizing Glycans on the Microscale for Microarray and Other Applicati

        互联网

        1605
        • Abstract
        • Table of Contents
        • Materials
        • Figures
        • Literature Cited

        Abstract

         

        Nature possesses an unlimited number and source of biologically relevant natural glycans, many of which are too complicated to synthesize in the laboratory. To capitalize on the naturally occurring plethora of glycans, a method is presented here to fluorescently tag isolated free glycans while maintaining the closed?ring structure. After purification of the labeled glycans, they can be printed on a glass surface to create a natural glycan microarray, suitable for interrogation with potential glycan?binding proteins. The derivatization of these natural glycans has vastly expanded the number of glycans available for functional studies. Curr. Protoc. Chem. Biol. 3:53?63 © 2011 by John Wiley & Sons, Inc.

        Keywords: fluorescence; reductive amination; glycan microarray; conjugation

             
         
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library

        Table of Contents

        • Introduction
        • Basic Protocol 1: Preparation of Closed‐Ring Glycan‐AEAB from Free Reducing Glycans
        • Alternate Protocol 1: Preparation of AEAB Conjugates Using Commercial Chemicals
        • Commentary
        • Literature Cited
        • Figures
             
         
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library

        Materials

        Basic Protocol 1: Preparation of Closed‐Ring Glycan‐AEAB from Free Reducing Glycans

          Materials
        • 0.05 to 1 mg free reducing glycan, lyophilized (e.g., Sigma‐Aldrich, V‐labs, Carbosynth)
        • Milli‐Q purified water (Millipore), or equivalent
        • Ammonium bicarbonate
        • 50% (v/v) acetonitrile, HPLC grade (Fisher Scientific)/10 mM ammonium bicarbonate
        • 10 mM ammonium bicarbonate
        • Sodium bicarbonate
        • Saturated sodium bicarbonate solution, ice‐cold
        • Acryloyl chloride (e.g., Sigma‐Aldrich)
        • Sodium borohydride (e.g., Sigma‐Aldrich), optional
        • Acetic acid, ACS grade (Fisher Scientific), optional
        • 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid (TFA), HPLC grade (Fisher Scientific)
        • Methanol
        • Ethanol
        • Ozone
        • Nitrogen gas
        • Methyl sulfide
        • 7:3 (v/v) dimethyl sulfoxide (DMSO), ACS grade (Fisher Scientific)/acetic acid
        • Sodium cyanoborohydride
        • 2‐(N ‐aminoethyl)‐amino benzamide (AEAB) hydrochloride (Song et al., )
        • Acetonitrile
        • 1% (v/v) trifluoroacetic acid (TFA), HPLC grade (Fisher Scientific)
        • 1.5‐ml screw‐cap polypropylene centrifuge tubes
        • 55°C and 65°C heating block or water bath
        • 150 mg, 300 mg, and 1 g carbograph solid phase extraction (SPE) columns (Alltech)
        • Rotary evaporator (e.g., SpeedVac, Thermo Scientific)
        • Lyophilizer
        • 15‐ml conical polypropylene centrifuge tubes
        • Porous graphitized carbon (PGC) analytical HPLC column (Thermo Scientific)
        • High‐performance liquid chromatography (HPLC) system

        Alternate Protocol 1: Preparation of AEAB Conjugates Using Commercial Chemicals

          Materials
        • 7:3 (v/v) dimethyl sulfoxide (DMSO)/acetic acid solution
        • p ‐nitrophenyl anthranilate, 98% (PNPA, Fisher Scientific)
        • Sodium cyanoborohydride, 95% (Sigma‐Aldrich)
        • 0.05 to 1 mg free reducing glycan, lyophilized (e.g., Sigma‐Aldrich, V‐labs, Carbosynth)
        • Acetonitrile
        • 1% (v/v) trifluoroacetic acid (TFA)
        • Milli‐Q purified water, or equivalent
        • Ethylenediamine solution: dissolve 100 µl ethylenediamine in 1 ml DMSO
        • 10% (v/v) acetic acid
        • 65°C heating block or water bath
        • C18 analytical column
        • High‐performance liquid chromatography (HPLC) system
        • Rotary evaporator (e.g., SpeedVac, Thermo Scientific)
        • Lyophilizer
        • Hypercarb (PGC) HPLC column
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library

        Figures

        •   Figure 1. The closed‐ring fluorescent conjugation of reducing glycans.
          View Image
        •   Figure 2. The preparation of AEAB conjugates or fluorescent neoglycoproteins by the reaction of PNPA derivatives with ethylenediamine or proteins, respectively.
          View Image
        •   Figure 3. Comparison of the binding of four different antibodies to open‐ring and closed‐ring AEAB conjugates on a glycan array. The arrays were printed using a piezo printer (Perkin‐Elmer) with open‐ and closed‐ring AEAB derivatives at 300 µM on NHS‐derivatized slides. Antibodies were applied to the glycan array at the concentrations indicated in the figure, and detected with appropriate fluorescently labeled secondary antibodies (Song et al., ). The x axis represents different glycans on the array by number, and the y axis represents the relative fluorescence units (RFU) detected on the microarray.
          View Image

        Videos

        Literature Cited

        Literature Cited
           Alvarez, R.A. and Blixt, O. 2006. Identification of ligand specificities for glycan‐binding proteins using glycan arrays. Methods Enzymol. 415:292‐310.
           Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. 1995. Nonselective and efficient fluorescent labeling of glycans using 2‐amino benzamide and anthranilic acid. Anal. Biochem. 230:229‐238.
           Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J., van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.H., and Paulson, J.C. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U.S.A. 101:17033‐17038.
           Cummings, R.D. 2009. The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5:1087‐1104.
           de Paz, J.L. and Seeberger, P.H. 2006. Recent advances in carbohydrate microarrays. QSAR Combin. Sci. 25:1027‐1032.
           Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., and Smith, F. 1951. A colorimetric method for the determination of sugars. Nature 168:167.
           Feizi, T. and Chai, W. 2004. Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell Biol. 5:582‐588.
           Horlacher, T. and Seeberger, P.H. 2008. Carbohydrate arrays as tools for research and diagnostics. Chem. Soc. Rev. 37:1414‐1422.
           Luyai, A., Lasanajak, Y., Smith, D.F., Cummings, R.D., and Song, X. 2009. Facile preparation of fluorescent neoglycoproteins using p‐nitrophenyl anthranilate as a heterobifunctional linker. Bioconjug. Chem. 20:1618‐1624.
           Manger, I.D., Rademacher, T.W., and Dwek, R.A. 1992. 1‐N‐glycyl beta‐oligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes. Biochemistry 31:10724‐10732.
           Paulson, J.C., Blixt, O., and Collins, B.E. 2006. Sweet spots in functional glycomics. Nat. Chem. Biol. 2:238‐248.
           Smith, D.F., Song, X., and Cummings, R.D. 2010. Use of glycan microarrays to explore specificity of glycan‐binding proteins. Methods Enzymol. 480:417‐444.
           Song, X., Lasanajak, Y., Rivera‐Marrero, C., Luyai, A., Willard, M., Smith, D.F., and Cummings, R.D. 2009a. Generation of a natural glycan microarray using 9‐fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag. Anal. Biochem. 395:151‐160.
           Song, X., Lasanajak, Y., Xia, B., Smith, D.F., and Cummings, R.D. 2009b. Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem. Biol. 4:741‐750.
           Song, X., Xia, B., Stowell, S.R., Lasanajak, Y., Smith, D.F., and Cummings, R.D. 2009c. Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16:36‐47.
           Song, X., Lasanajak, Y., Xia, B., Heimburg‐Molinaro, J., Rhea, J.M., Ju, H., Zhao, C., Molinaro, R.J., Cummings, R.D., and Smith, D.F. 2011. Shotgun glycomics: A microarray strategy for functional glycomics. Nat. Methods 8:85‐90.
           Stevens, J., Blixt, O., Paulson, J.C., and Wilson, I.A. 2006. Glycan microarray technologies: Tools to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857‐864.
           Stowell, S.R., Arthur, C.M., Dias‐Baruffi, M., Rodrigues, L.C., Gourdine, J.P., Heimburg‐Molinaro, J., Ju, T., Molinaro, R.J., Rivera‐Marrero, C., Xia, B., Smith, D.F., and Cummings, R.D. 2010. Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16:295‐301.
           Xia, B., Kawar, Z.S., Ju, T., Alvarez, R.A., Sachdev, G.P., and Cummings, R.D. 2005. Versatile fluorescent derivatization of glycans for glycomic analysis. Nat. Methods 2:845‐850.
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library
         
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序