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pro-collagen extraction from Mouse Tissues

protein extraction procollagen

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#1 k.g.

k.g.

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Posted 30 April 2012 - 08:54 AM

Hi, new to the site. I am currently trying to extract procollagen from mouse organs, kidney and liver mostly.

I originally used a buffer with extraction buffer of
-20mM Tric-HCl
-150mM NaCl,
-1mM EDTA,
-1% Triton X-100.
On top of that I add 40ul Protease inhibitor cocktail (sigma) to every 1ml of extract buffer right before I use the buffer for extraction. This is a buffer that I used in the past and have success extracted other proteins with it but when I tried it to extract pro-collagen I got really poor results. I read the samples in an ELISA and had to use 10mg/ml total protein to get any reaction, which was, still very week and did not make it onto the curve. I can typically use 5mg/ml of total protein for the other proteins that I extract which I are in far smaller quantities in the cell than collagen and procollagen. I know that collagen is insoluble and this is probably why I did not recover very much procollagen.

Does any body have any experience with extracting procollagen so that it can be run in an ELISA or how to extract Collagen to be run in an ELISA?

Thanks!

Edited by k.g., 30 April 2012 - 11:01 AM.






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