Hello everybody,
I'm preparing stock aliquots of cell culture supplements, and I have a problem dissolving insulin. The old protocol in my lab that I tried to use first says to dissolve insulin in sterile water which, as I found from experience, is just plain wrong because insulin has very poor solubility at neutral pH.
Most other protocols I found recommend to dissolve insulin in acidized water with pH 2-3 (0.005 N HCl). But some protocols also say that once it's dissolved, the pH has to be reconstituted to neutral apparently because acid will eventually destroy insulin molecules.
To be honest, I have no desire to adjust solution pH with pH meter because I want to prepare the solution aseptically without removing it from the cell culture hood. Some people also claim that insulin would form precipitate again if the solution is neutralized. So what if I just froze the insulin aliquots in acidized water? Will it be preserved, or the acid is destructive even in the freezer?
When I will eventually thaw and use the aliquots they will be diluted about 200-300 times with buffered cell culture medium, so I guess the acid would be too diluted to present any harm for cells.
Do you have any experience with it?
Hello, at low pH human insulin is known to degrade at the C-terminal Asnl by both deamidation and covalent dimerization. Also, it is known that deamidation at internal Asn residues can occur through intramolecular catalysis via a reactive cyclic imide intermediate which further hydrolyses to Asp.
So avoid low pH 2-5 for storage and use 6.0-7.5 for more stability.
Hope this info will help you.
Good luck!
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