Hi Amir,

trypsin is not so effcient for ECM digestion, you could try other enzyme likewise Dispase or Ialuronidase.

Good luck

Great thanks Dr.  Cesare for your reply. I will try to use disease, but can I use it with the same concentration as pepsin, or there will be differences?

Amir:

the collagens consist of assembled triplehelical molecules. Famously, collagen triplehelices are pepsin and trypsin stable ! That is why you use pepsin to lift intact triplehelices out of tisue and when you neutralise the triplehelices can still rearrange to form fibrils. Every triplehelix has two non triplehelical ends, the telopeptides. Via these ends the triplehelixes get more stabil crosslinked after fibril formation. When pepsin comes in, this will destry the outer portions of the telopeptides, and this is how you partiall destry teh crosslinks. So you can lift out single triplelhelices or packeges of them out of tissue witouth destroying the triplehelices as such. You need them later for hydrogel formation. This is how the hydrogel forms. Only if the triplehelix is partially unfolded, pepsin or trypsin can get in edgewise and rip the area apart. Of course, if you just digest for long enough damage will occur to triplehelices, as well. In your case an enyzme that might play a similar role as pepsin is chymotrypsin. Trypsin as such will not attack the triplehelices and might not be efficient in attacking the telopeptides, either. Details can be foudn in the literature and data sheets of the enzymes. Hyaluronidase will , as the name suggest only attack hyaluronic acid. Dispase  cleaves collagen to a limited extent, but cant tell where exactly. However, teh Worthington manual gives you valuable info on which amino acids the enyzme cleaves and then you can look up whether there are these cleavage sites in telopeptides of collagen I, the most abundant collagen type in most tissues. Cheers , Michael

Great thanks, prof. Michael for your reply

From your explanation and the details of specificity and working conditions of the candidate enzymes, it seems that the usage of an alternative to pepsin needs standardization of different factors including the concentration and the incubation time with the ECM powder. I will check the optimum conditions for the action of chemotrypsin or dispase if possible, and hope one of them will succeed.  

Dear Amir,

Have you checked the optimum conditions for chemotrypsin, dispase or trypsin?

I'm looking for other enzymes to digest ECM.

Amir Mohammed Alsharabasy

Hi Ahed

I tried to use trypsin instead of pepsin, but the digestion was very weak (or negligible), but finally I used pepsin.

Hi Amir,

I would like to know what your pepsin concentration was?

10 mg of pepsin for 100 mg dECM?

How long does it take to have uniform gel?

Did you keep your gel below 10 °C?

Thank you in advance.

Amir Mohammed Alsharabasy