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High-Specific-Activity 35S-Labeled Heparan Sulfate Prepared from Cultured Cells

  • Nicholas W. Shworak
Part of the Methods in Molecular Biology™ book series (MIMB, volume 171)

Abstract

Cultured cells are a facile reagent for elucidating the molecular mechanisms that regulate the biosynthesis of heparan sulfate (HS) (1, 2, 3). However, a typical confluent flask (∼20 million cells) produces only a small amount of HS (1–2 µg), which is at or below the detection limit of many nonradioisotopic techniques. Fortunately, this limitation can be circumvented by the metabolic labeling of cells with Na2 35SO4. Sulfate from the culture medium is transported into the cytoplasm, where it is incorporated into the biosynthetic sulfate donor adenosine 3′-phosphate 5′-phosphosulfate (PAPS), which is transported into the Golgi apparatus (4). Specific biosynthetic enzymes transfer a sulfonyl group from PAPS onto maturing glycosaminoglycan chains.

Keywords

Heparan Sulfate Equilibration Buffer Radioactive Contamination Metabolic Label Label Medium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Nicholas W. Shworak
    • 1
  1. 1.Department of MedicineHarvard Medical School; Angiogenesis Research Center, Beth Israel Deaconess Medical CenterBoston

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