What you wish you knew before storing your protein in the freezer

Storing protein samples sounds like an easy thing to do in the lab. After all, you just put them in the freezer right? But is that all to it? Can all proteins withstand freezing (and thawing) without losing their function? How long can you store a protein before it loses its function? What could be added or removed to extend their shelf-life?

Contrary to what many researchers may think, there is no universal storage practice that can be applied to all proteins that ensure their integrity will indefinitely be maintained.

In fact, proteins can be quite picky and each one has its own preferences. In order to understand the different ways proteins can be stored, here are a few tips and suggestions.

Storage time dictates how cold proteins should be kept

Proteins can be stored at 4°C if they’re going to be used within a few days. However, if you’re going to keep your proteins at 4°C, make sure they’re in clean, autoclaved tubes since they’re susceptible to microbial and proteolytic degradation at this temperature. You can also add antimicrobial agents such as sodium azide or thimerosal. If proteolysis becomes a problem, consider adding protease inhibitor cocktails as it can help. This is especially important when storing either serum preparations or ascitic fluid since both contain proteases.

For longer storage periods, samples can be stored frozen or in solution at -20°C. While freezing allows for a longer shelf-life (few years), samples should be removed only once from the freezer since repeated freeze-thaw cycles can cause protein degradation. On the other hand, while storing samples in solution reduces their shelf-life (~1 year), it will allow you to use them repeatedly from a single stock. The trick here is to store your proteins with cryoprotectants that prevent the formation of ice crystals, which can destroy protein structure. You can also dialyze your sample against the storage buffer containing the cryoprotectant. This alternative can result in a more concentrated and stable protein.

If you need your protein samples for even longer periods of time (several years), storing them at -80°C or even in liquid nitrogen are your best options. At this temperature, protein samples are less likely to get contaminated so there’s no need to add antimicrobials.

It’s best to store proteins in a more concentrated form

In general, diluted proteins (less than 1 mg/ml) are more prone to inactivation. Therefore, it is highly recommended to store your proteins at high concentrations — at least 1 mg/ml. If you can’t reach this level, you can add a “filler” protein such as bovine serum albumin to reach a greater concentration. Also, the more concentrated your protein solution is, the less you’ll lose from it binding to the storage tube walls.

Additives can extend your protein’s shelf life

In addition to the already mentioned cryoprotectants, polymers like polyethylene glycol and polysorbates such as Tween 80 can be used to extend your protein’s shelf life. You can also consider adding metal chelators — such as EDTA — and reducing agents — such as DTT — to avoid metal-induced oxidation and maintain a protein’s reduced state.

So the next time you need to store your protein, make sure you follow these tips to ensure you don’t lose your protein to the freezer.