Isolation of proteoglycans using phenol extraction and isopycnic centrifugation in cesium trifluoroacetate.
A procedure for the rapid isolation of heparan sulfate proteoglycans from Zwittergent lysates of cultured cells and cetylpyridinium precipitates of culture medium is described. After removal of most nonglycosylated proteins by phenol extraction, the proteoglycans are fractionated and isolated by isopycnic sedimentation in cesium trifluoroacetate gradients. The density-isolated proteoglycans, which are largely free of high molecular weight DNA, RNA, and nonglycosylated proteins, are readily amenable to chemical and enzymatic characterization. These procedures, which are designed for efficient and rapid isolation of lipophilic proteoglycans, also appear applicable to the isolation of other high density proteoglycans.
McBain
J A
JA
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Mueller
G C
GC
D002500:Centrifugation, Isopycnic; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D003110:Colonic Neoplasms / Q000378:metabolism; D003470:Culture Media / Q000032:analysis; D003902:Detergents; D010450:Endopeptidases; D006493:Heparin / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D010636:Phenols; D011509:Proteoglycans / Q000302:isolation & purification*; D014269:Trifluoroacetic Acid; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003470:Culture Media; D003902:Detergents; D007202:Indicators and Reagents; D010636:Phenols; D011509:Proteoglycans; D006493:Heparin; D014269:Trifluoroacetic Acid; D010450:Endopeptidases