We are used the protocol for urease extration adaptated (to C. neoformans) from Amin et al.,2013:
Briefly, the broth cultures were subjected to centrifugation (5,000 × g, 4 °C) and the recovered mass was washed twice using phosphate-buffered saline (pH 7.4) and then stored at -80 °C. Subsequently, was thawed to ambient (room) temperature, followed by mixing with 3 mL of distilled water and protease inhibitors (TLCK, Tosyl-L-lysyl-chloromethane hydrochloride) and sonication for 60 s. After centrifugation (15,000 × g, 4 °C), the supernatant was desalted by eluting through SephadexG-25 column. The resultant crude urease solution was mixed with an equal volume of glycerol and then preserved under refrigerator (4 °C) for further uses.
Okay, we ran the suggested protocol, but after 2 days, enzyme activity was no longer observed.
Then, we tried to exclude the TLCK from the protocol, again the activity was observed on the day of extraction, but after it lost the activity.
We also tried to activate urease with sodium bicarbonate and Ni solution, without success.
In another procedure, we repeated the protocol and lyophilized the solution resulting in a white solid. So we tested it on this day, we prepared a urease solution (10 mg / mL) and the activity was good, but after a few days, again the activity was lost.
We always work with refrigeration, and we are careful to keep the solution on ice when we handle it.
Please any suggestions?
You can read: Article Cryptococcus gattii urease as a virulence factor and relevan...
Something I would have done differently is to have used a pH-buffered solution during the procedure to maintain the pH at the optimal pH of the enzyme.
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