May be your buffer solutions are contaminated with proteases...The D/W you use should be free of proteases..You can also do a bradford assay at each step of your protocol to check the concentration of proteins..

Thanks a lot for your reply. Do you think collagen can be that easily digested by proteases? I am using DDW. As for your second suggestion, I will certainly do it this week... Concerning my collagen control, do you have any idea what could be the issue? I was told that the collagen I used may be too much crossed-linked and thus, pepsin would not be enough to dissolve it. but then, I used collagenase...and there was no band either...

Shouldn't collagenase digest the collagen? How could you detect it then? If your control is negative then you have probably problem with detection. How do you detect the proteins? Only with Coomassie? Are you sure it binds to collagen? I think it binds to either aromatic or positively charged amino acids, neither of which is present in collagen to my knowledge.

Dear Tomas, thank you for your answer. you are right, collagenase does digest collagen. but as I am using a too crosslinked collagen, I thought reducing the crosslink content. I used only coomassie to detect these proteins. It has been widely reported already in scientific papers that you can detect collagen from cell extracts by simply harvesting the cells (or medium), followed by pepsin digestion and SDS-PAGE with coomassie blue staining. That is why I am really getting frustrated over this as I really don't know what could have gone wrong...

Did your sample get to hot on sonication?

Dear Steven, it was indeed slightly hot. I normally put the samples in a glass beaker with 4°C cold water.

I normally use a little ice as our sonicatior is really old and I found my yeilds were really low probably due to denaturing. I tend to use shop bought lysis buffers instead as I get better yeilds.

But the collagenase will digest the collagen, not the cross-links. How is it cross-linked?

Do you know an approx size of your cross linked collagen?

I have a question, couldn't you just add Chelating agents like EDTA and EGTA which would remove Calcium ions from Collagenase and thereby inhibit it? Just a thought.

Hi all, Collagen in bovine tendon achille is highly cross-linked and very insoluble in water due to post-translation modifications. I unfortunately don't know the size of the cross-links. Do you think Urea could help solubilize it? thanks again for all your comments.