Temperature wise, I'd recommend flash freezing small aliquots for single use in liquid nitrogen and storing them at -80 or lower (long term at -130 or below).

However, you need to adapt the freezing buffer for enzyme activity survival, possibly by making it isotonic, optimize pH, maybe adding reductants and scavengers like DTT, etc.).

Another option could be freeze drying with a so-called glass buffer and storage in the fridge or lower.

In any case, however, you need to demonstrate the viability (and the influence of variables) of your method of choice.

Hey, I freeze aliquots of conditioned medium at -20 with 3 months viability. I also work with metalloproteases and it worked perfectly for zymography. As long as you are not interested in the RNA content of the medium, it can be frozen at -20

Vatamanu Sergiu-Marian Hi, I am trying to do zymography and use a lysis buffer with Tris, NaCl, and NP-40, but the sample stays very viscous making it very difficult to load on the gel. How should I process it to use so that the enzyme activity is also retained? Do you have any suggestions?

Thank you