Type I collagen - extraction and purification - (Jan/16/2006 )

I was wondering if anyone has working experience with extracting and purifying type I collagen from mouse?

I am looking for a simple protocol.

I am interested in the propeptide, not the mature processed collagen I.

I have both tissue (bone, skin) and fibroblast cell culture to work with, although tissue is difficult since the turnover rate to mature collagen is quite fast and hard to control.

Has anyone tried the simple RIPA cell lysate/media extraction method? Most protocols resort, first, to acid extraction, then neutralization, then precipitation, then dialysis, then column elution, then another round of solubolization, then who knows what has happened to the protein by now?
What is important is plenty of protease inhibitors in order to prevent the conversion to mature collagen within the solutions

Once again, does anyone have real hands on experience with working on collagen type I protein?

any assistance will be of great help

sincerely,

thomas

-anyway_night-

QUOTE (anyway_night @ Jan 16 2006, 04:21 PM)

hello Thomas, I may be abit late, but check out this paper: Ruangpanit et al, 2001: Gelatinase A activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form. It contains a good method for isolating type I collagen.
The acid extraction will extract the procollagen form. you can adapt this protocol for mouse skin.

-haison-