Every time I conduct protein crystallization experiments, it takes a considerable amount of time, often extending into the late hours of the night. Therefore, I am considering flash-freezing the purified protein samples in liquid nitrogen and storing them in a -80°C freezer. The idea is to thaw them the next day and continue the experiments. Is this approach viable?
The issue is that glycerol is an antinucleating agent. Because of this, glycerol prevents ice damage when freezing proteins, but also potently poisons crystal nucleation because it inhibits protein aggregation which is required for crystal growth. Dialysis can actually not be efficient in removing enough glycerol. But if you have already been able to crystallize a protein from a glycerol containing buffer then maybe you don't have much to worry about.
A crystal structure tells you surprisingly little since AlphaFold2 came out. Does the electron density correspond to a water molecule or glycerol or a molecule of MES buffer, or maybe ligand? Enzyme kinetics of deletion mutants and point mutation mutants tells you a lot. A crystallographically disordered region could be absolutely essential for catalysis.
The critical point in freezing proteins for storage are the freezing and thawing processes. While freezing is a good solution for long-time storage, I would avoid repeatedly freezing and thawing the protein just to avoid keeping it for a few hours at 4° C. For the same reason, if you need the same protein prep for multiple experiments, it is better to aliquot the stock solution before freezing it, rather than repeatedly thawing the stock just to take out a small fraction, then freezing it again.
Of course, it very much depends on the specific protein and other components of the sample how much damage can be done by either storage at 4° or by freezing/thawing.
You can try this.
There are two challenges that your proteins will face: Freeze and thaw shock and ultimately, reduced (native) yield.
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