Purification of fibrillin-containing microfibrils and collagen VI microfibrils by density gradient centrifugation

Analytical Biochemistry
C M KieltyC A Shuttleworth

Abstract

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M(r) microfibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS-PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

References

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Jan 1, 1983·International Review of Connective Tissue Research·E G Cleary, M A Gibson
Aug 1, 1995·The International Journal of Biochemistry & Cell Biology·C M Kielty, C A Shuttleworth
Mar 1, 1994·The Journal of Cell Biology·C M Kielty, C A Shuttleworth
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Citations

Jun 9, 2014·Human Molecular Genetics·Tadashi InoueTomoyuki Nakamura
Jan 24, 2007·The Journal of Cell Biology·Shazia S ChaudhryCay M Kielty
Apr 13, 2010·Matrix Biology : Journal of the International Society for Matrix Biology·Mahroo K ParsiMark A Gibson
Feb 13, 2007·Matrix Biology : Journal of the International Society for Matrix Biology·Rena HiraniMark A Gibson
Apr 30, 2008·Methods : a Companion to Methods in Enzymology·Stuart A CainCay M Kielty
Feb 18, 2018·The Journal of Biological Chemistry·Alexander EckersleyMichael J Sherratt
Nov 23, 2005·Proteomics·Stuart A CainCay M Kielty
Dec 13, 2006·The Journal of Biological Chemistry·Chiu-Liang KuoLynn Y Sakai
Aug 11, 2000·Micron : the International Research and Review Journal for Microscopy·M J SherrattC M Kielty

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