Will non-denatured proteins degrade more easily? How long can extracted proteins be stored at -80°C?
Two aspects need to be distinguished: the impact of protein's inherent structural state and storage conditions on stability.
I. Degradation risk of native proteins
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Proteins in their native conformation retain their complete spatial structure and usually remain active. If residual proteases or trace metal ions are present in the solution, these proteins may be easily recognized and cleaved, leading to rapid degradation.
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Denaturing treatments (such as adding SDS, urea, or heating) will result in loss of structure and activity, making it difficult for most proteases to cleave them. However, the denatured state is usually unsuitable for functional assays or structural studies.
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Therefore, native proteins require stringent inhibition of protease activity (by adding a protease inhibitor cocktail) and should be kept at low temperatures.
II. Storage period at -80°C
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Proteins can be stably stored at -80°C for several months to a year when glycerol (commonly 10–50%) or a small amount of buffering salts are present, depending on the inherent stability of the protein (such as structural complexity, disulfide bonds, whether it is a membrane protein, etc.).
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Without protective agents, when stored only in PBS or aqueous solution, freeze-thaw cycles can severely affect the structure, typically leading to a noticeable decline in activity within weeks to 1–2 months.
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Avoid frequent freeze-thaw cycles: it is recommended to aliquot into small volumes for single-use.
Recommendation
Determine the type of protein and its subsequent use (activity assay, structural study, or mass spectrometry analysis), choose the appropriate buffer system and protective agents, and add a broad-spectrum protease inhibitor during preparation to significantly extend the storage period.
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