collagen extraction

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parthy
parthy's picture
collagen extraction

All;

I am looking for a protocol to extract soluble collagen from mouse kidney tissue and measure it via a calorimetic assay (Sirius red - dye binding assay)

Any one has a detailed  protocol for collagen extraction by acid- pepsin digestion?

Thanks
parthy

The FFM
The FFM's picture
The following paper contains

The following paper contains a protocol for the isolation of collagen using  acetic acid and pepsin

Matrix Biol. 2001 Jun;20(3):193-203.
Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form.
Ruangpanit N, Chan D, Holmbeck K, Birkedal-Hansen H, Polarek J, Yang C, Bateman JF, Thompson EW.
VBCRC Breast Cancer Invasion and Metastasis Unit, St. Vincent's Institute of Medical Research, 9 Princes St., 3065, Fitzroy, Australia.

Abstract
The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 microg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.
 

PMID: 11420151 [PubMed - indexed for MEDLINE]