I am working with a protein that degrades over time, and I am looking for an optimal method to preserve its activity for later analysis. The protein is secreted into a marine-based medium, and it is sensitive to temperature, freezing, and prolonged storage.
So far, I am considering using 50 mM Tris-HCl (pH 7.5) with 10% glycerol as a potential preservation buffer. However, my main concerns are about Preventing loss of protein activity over storage. also Ensuring that the storage buffer is compatible with size-exclusion chromatography (SEC). And Which additional stabilizers might improve preservation without interfering with SEC.
I would appreciate any recommendations on:
Optimal buffer conditions for protein stability Best storage temperature (-20°C, 4°C, or liquid nitrogen?)
Whether flash freezing or lyophilization would be better than glycerol storage Any practical tips to prevent degradation while maintaining SEC compatibility
Thanks in advance for your help!
Flash-freezing in small aliquots using liquid nitrogen, if it is available, is probably the best freezing method. If liquid nitrogen is not available, then a dry-ice ethanol bath can be used. Including 10% glycerol is a good idea. Another often useful stabilizing agent is the sugar trehalose, at least 0.5 M. Store the frozen protein at -80oC or in liquid nitrogen. Avoid lyophilization.
There is no way to predict "the best" storage conditions for an unknown protein. You need to test if your protein can survive one or multiple freeze-thaw cycles (with or without glycerol or other stabilizers, slow or fast freezing, etc). If yes, usually storage at -80 is a safe way to store in small aliquots, or -20 but again, for long-term storage even -20 could show some unpleasant surprises.
Buffer pH, with or without salt like NaCl or divalent cations like Mg or Ca, all of these could have an influence.
As a rule of thumb, standard PBS buffer is probably the worst buffer for frozen storage but still widely used. I would try to avoid phosphate buffers.
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