Purification of fibrillin-containing microfibrils and collagen VI microfibrils by density gradient centrifugation.
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.
Kielty
C M
CM
School of Biological Sciences, University of Manchester, United Kingdom. cay.kielty@man.ac.uk
Hanssen
E
E
Shuttleworth
C A
CA
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification*; D003238:Connective Tissue / Q000737:chemistry / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D000071837:Fibrillins; D015151:Immunoblotting; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008854:Microscopy, Electron; D012967:Sodium Dodecyl Sulfate; D013329:Structure-Activity Relationship
D000071837:Fibrillins; D008840:Microfilament Proteins; D012967:Sodium Dodecyl Sulfate; D003094:Collagen