• 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

        Metabolic Labeling with Sulfate

        互联网

        793
        • Abstract
        • Table of Contents
        • Materials
        • Literature Cited

        Abstract

         

        Post?translational modifications of proteins make it possible to determine where a protein normally resides or to follow its transport through the cell. One such modification is addition of sulfate either to tyrosine residues or to carbohydrate side chains. Labeling studies with [35 S] sulfate can be done as continuous or pulse?chase experiments, as described here.

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

        Table of Contents

        • Basic Protocol 1: Long‐Term [35S]Sulfate Labeling of Monolayer Cells in Culture
        • Basic Protocol 2: Pulse‐Chase [35S]Sulfate Labeling of Monolayer Cells in Culture
        • Reagents and Solutions
        • Commentary
             
         
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library

        Materials

        Basic Protocol 1: Long‐Term [35S]Sulfate Labeling of Monolayer Cells in Culture

          Materials
        • Dialyzed serum (see recipe )
        • 200 mM L‐glutamine in H 2 O
        • Sulfate‐free DMEM (see recipe )
        • Sodium [35 S]sulfate (>1000 Ci/mmol)
        • Cells of interest, cultured to 70% to 80% confluence
        • DPBS ( appendix 2A ), 4°C
        • Additional reagents and equipment for immunoprecipitation of proteins (unit 7.2 ) or for SDS‐PAGE (unit 6.1 ) and fluorography (unit 6.3 )
        NOTE: The labeling medium is a modified formula for DMEM, which the author has found works best. Other media, such as RPMI, have not been used as successfully.NOTE : All solutions for overnight labeling should be prepared and used under aseptic conditions.NOTE : The size of the cultures used depends on the amount of the protein of interest present in the cells and the techniques used for subsequent analysis.

        Basic Protocol 2: Pulse‐Chase [35S]Sulfate Labeling of Monolayer Cells in Culture

          Materials
        • Cultured cells of interest, ∼70% to 80% confluent
        • Dialyzed serum (see recipe )
        • 200 mM L‐glutamine in H 2 O
        • Sulfate‐free DMEM (see recipe )
        • Sodium [35 S]sulfate (>1000 Ci/mmol)
        • DPBS ( appendix 2A ), 4°C
        • Chase medium (see recipe )
        • Platform rocker
        • Additional reagents and equipment for immunoprecipitation of proteins (unit 7.2 ), SDS‐PAGE (unit 6.1 ), and fluorography (unit 6.3 )
        GO TO THE FULL PROTOCOL:
        PDF or HTML at Wiley Online Library

        Figures

        Videos

        Literature Cited

        Literature Cited
           Baeuerle, P.A. and Huttner, W.B. 1986. Chlorate—A potent inhibitor of protein sulfation in intact cells. Biochem. Biophys. Res. Commun. 141:870‐877.
           Beisswanger, R., Corbeil, D., Vannier, C., Thiele, C., Dohrmann, U., Kellner, R., Ashman, K., Niehrs, C., and Huttner, W.B. 1998. Existence of distinct tyrosylprotein sulfotransferase genes: Molecular characterization of tyrosylprotein sulfotransferase‐2. Proc. Natl. Acad. Sci. U.S.A. 95:11134‐11139.
           Bowman, K.G. and Bertozzi, C.R. 1999. Carbohydrate sulfotransferases: Mediators of extracellular communication. Chem Biol. 6:R9‐R22.
           Farzan, M., Mizabek, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N.P., Gerard, C., Sodroski, J., and Choe, H. 1999. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV‐1 entry. Cell 96:667‐676.
           Huttner, W.B. 1984. Determination and occurrence of tyrosine O‐sulfate in proteins. Methods Enzymol. 107:200‐223.
           Huttner, W.B. and Baeuerle, P.A. 1988. Protein sulfation on tyrosine. In Modern Cell Biology, Vol. 6 (B. Satir, ed.) pp. 97‐140. Alan R. Liss, New York.
           Linhardt, R.J. 1994. Analysis of glycosaminoglycans with polysaccharide lyases. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.C. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 17.13.17‐17.13.32. John Wiley & Sons, New York.
           Niehrs, C. and Huttner, W.B. 1990. Purification and characterization of tyrosylprotein sulfotransferase. EMBO J. 9:35‐42.
           Niehrs, C., Huttner, W.B., and Rüther, U. 1992. In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites. J. Biol. Chem. 267:15938‐15942.
           Yanigashita, M., Salustri, A., and Hascall, V.C. 1989. Specific activity of radiolabeled hexosamines in metabolic labeling experiments. Methods Enzymol. 179:435‐445.
        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