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Metabolic Labeling with Sulfate

关键词： metabolic labeling sulfate来源：互联网

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.

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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

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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 )

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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.

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