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96-well plate assays for measuring collagenase activity using 3H-acetylated collagen

Lookup NU author(s): Paul Koshy, Emeritus Professor Drew Rowan, Emeritus Professor Tim Cawston


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We describe two alternative assays for measuring collagenolytic activity using H-3-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble 3H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity greater than or equal to 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added. (C) 1999 Academic Press.

Publication metadata

Author(s): Koshy PJT, Rowan AD, Life PF, Cawston TE

Publication type: Article

Publication status: Published

Journal: Analytical Biochemistry

Year: 1999

Volume: 275

Issue: 2

Pages: 202-207

Print publication date: 01/11/1999

ISSN (print): 0003-2697

ISSN (electronic): 1096-0309

Publisher: Academic Press


DOI: 10.1006/abio.1999.4310


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