I obtained ECM from cardiac tissue (I don't have much sample) and I want to make a solution of this ECM to be able to make a coating for cellular culture. Does anybody know how can I homogenize the ECM in the solution in order to have a solution without ECM clumps?
Thank you in advance.
You can try dissolving it in acetic acid, but the traces of acetic acid should be removed before you extract the ECM back.
I have a protocol that digest the ECM in a 1:10 Pepsin in 0,1M HCl solution for 48h and then dilutes the ECM solution with 0,2M Acetic acid. But after that I still have ECM clumps
The protocol you have is likely quite suitable to "solubilize" the ECM. Nevertheless according to the animal species there will be always a variable and even very significant age-related amount of insoluble material. You therefore could separate the insoluble material by centrifugation or alternatively make a fine suspension of the original whole ECM by repeated sonication.
Since I would like to seed some cells on top and the evaluate their behaviour, I assume that sonication is not damaging the ECM protein function.
On the other hand, when separating the insoluble material from the soluble one by centrifugation, Won't I be loosing some particular component of te ECM?
Thanks for the contribution.
In case you can also anlyse apart what you are loosing. However even ultrasonication is not fully impact free (mind thermal effects). Take also into account that among the soluble components obtained after pepsin treatment the triple helical domain of collagen will be the almost only preserved component. Pepsin is a powerful endopeptidase that wont spare almost any other protein. Unfortunately there are no easy shortcuts in the investigation of a multicomponent material like the ECM resulting from million years of evolution.
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