By this method, dilution of the specimen was
avoided and cleaner solutions were available for analyses. Beside simplicity and rapidity, this
procedure is found to be an effective method in complex sample protein separation [94–96] and it was
used here for the first time for whole animal tissues. We found the phase separation method to be a
very convenient approach, which allowed separating over the 90% of the proteins present in the
extract. The phase equilibrium of ACN-water systems after the phase separation showed that the
bottom phases (IP and LP) contain a certain amount of ACN, since they did not freeze at −20 °C [100].
This makes this phase separation method more attractive for bioactive molecules, since no strong
organic solvent is used and no damage of freeze-sensitive proteins occurs in presence of the
highly polar solvent ACN. We routinely used this method in our protocol before the samples were
lyophilized and we propose this method for the extraction of bioactive molecules from marine
gelatinous organisms.