By Renato V. Iozzo
Renato Iozzo and an authoritative workforce of investigators current for the 1st time a accomplished and updated number of conveniently reproducible preparative and analytical equipment for the in-depth research of those very important compounds. that includes step by step protocols, this booklet will let either beginner and skilled researchers to isolate intact proteoglycans from tissues and cultured cells, to set up the composition in their carbohydrate moieties, and to generate concepts for prokaryotic and eukaryotic expression. There also are targeted thoughts for the suppression of particular proteoglycan genes, for the detection of mutant cells and their degradation items, and for learning particular interactions among proteoglycans and extracellular matrix proteins and with progress elements and their receptors.
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Extra resources for Proteoglycan Protocols (Methods in Molecular Biology)
Example text
Perlecan from Endothelial Cells 27 4 Purification of Perlecan from Endothelial Cells John Whitelock 1. Introduction Perlecan, the major heparan sulfate (HS) proteoglycan of basement membranes and other connective tissues, is a modular molecule with five structural domains. It has been isolated from various sources including kidney and placenta, but most commonly from the mouse Englebroth Holm Swarm (EHS) tumor (1,2). It has also been purified from cultured cell lines such as endothelial cells (3) or fibroblasts (4).
The basic method involves preliminary extraction in nondemineralizing denaturing buffers, followed by extraction with demineralizing and denaturing solutions combined. The denaturing buffer extract is considered to be the osteoid-associated proteoglycan pool, while the demineralizing buffer extract is taken as the mineral-associated pool of proteoglycans. Following extraction procedures, the samples are subjected to anionexchange chromatography and size-exclusion chromatography for purification.
Tendon is difficult to powder. To avoid gumming up the mill, it is necessary to keep the grinding surfaces cold by adding liquid nitrogen continually. Some loss of tissue is inevitable. For smaller amounts of tissue one can use a tissue macerator (a shaking steel ball and chamber) cooled with liquid nitrogen. 10. The ratio of extraction fluid to tissue can be varied, depending on the goal of the extraction. Tensile tendon swells a great deal during extraction, whereas compressed tendon and cartilage do not.