We are extracting type 1 collagen through a base, HCl, salt precipitation, and freeze drying method and have noticed that the extracted protein looks like glass. Well at least glossy, and the fibers are not compact. We have tried concentrating our gels before lyophilizing, different freezing temperatures, using acetic acid instead of HCl, and pressing the collagen at up to 8 tons. Our SDS-PAGE shows that the same banding patterns appear (alpha [1,2], beta, and gamma bands) as Sigma-Aldrich rat tail derived type 1 collagen, but our bands are all slightly shifted up 5-10 KDa. What is causing this glassy looking collagen fiber formation. Thank you for viewing and any input is appreciated!
Hi Ian
When completing your salt precipitation did you remove the excess salts through some method like dialysis? Previously when I've done salt precipitation using ammonium bicarbonate or ammounium sulphate excess salts will precipitate out of solution and form crystals similar to the ones in your photo.
Proteins can also bind to the salts and form crosslinks which will give you these fiberglass structures.
Cheers,
Euan
Thank you Euan for the response! We dialyze VS DI water in a cellulose membrane 12000-14000 Da pore for about 2 days with bath changes twice a day. I'm going to try around 5 days to see how that works and update the thread.
Hi Ian,
you could also try to freeze-dry twice or three times. For some of our materials such as PEG-peptide conjugates, the material gets "fluffy" only after several freeze-drying steps. Do not use ultrasonication to re-dissolve, this will destroy the protein structure. I did it once and no fibrillar network could be formed with that sample any more performing a reconstitution protocol.
Hi Marcus,
When you freeze dry a second time, do you dissolve your protein in acid or just wet it in water?
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