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Collagen bands in Western Blot - (Jun/30/2008 )

Hello!! I have a problem. When I measure collagen type I by Western Blot in human tissue samples(heart), I can see one band of around 90 kDa, but when we measure collagen type I by Western Blot in in vitro experiments with cardiac fibroblasts we observe three specific bands:one of 90kDa, one around 110 kDa and one around 135 kDa. We think that these three bands could correspond to different forms of collagen type I. We have realyzed a MALDI analysis of these three bands, but we have a lot of problems to identify them.
Have anyone had a similar problem?

-JaviPPAR-

QUOTE (JaviPPAR @ Jun 30 2008, 01:00 PM)
Hello!! I have a problem. When I measure collagen type I by Western Blot in human tissue samples(heart), I can see one band of around 90 kDa, but when we measure collagen type I by Western Blot in in vitro experiments with cardiac fibroblasts we observe three specific bands:one of 90kDa, one around 110 kDa and one around 135 kDa. We think that these three bands could correspond to different forms of collagen type I. We have realyzed a MALDI analysis of these three bands, but we have a lot of problems to identify them.
Have anyone had a similar problem?


Are you sure the band from the tissue samples is 90kDA?
Monomeric collagen type I consists of alpha1 and alpha2 chains. The alpha 1 is 139kDa and the alpha2 is 129kDa. One collagen type I triple helix consists of 1 alpha2 chain and two alpha1 chains. When they are cross-linked, and these cross-links you cannot break by denaturing samples for SDS-PAGE, you also get the dimeric (about 270 kDa) and trimeric form (about 400 kDa). Off course it is hard to really see the size of these large fragments on SDS-Page because the separation capacity of the gels is not very good in this range, so you can see a 130 kDa fragment with two fragments above (the dimeric and trimeric forms). That is why I ask you if you are sure that the fragment is 90 kDa, because that than might be a degradation product or something or no collagen at all.

-aspergillie-

wrong subforum - please a moderator move this topic. mellow.gif

-ph3no-