Like graduate students, proteins are sensitive to rough handling. This is particularly true when they (the proteins, not the students!) are being concentrated, purified, and stored. We’ve covered the many options out there for concentrating your proteins, along with how to handle protein extracts to keep your proteins safe from degradation.
But proteins can degrade even after you’ve extracted and concentrated them! Storage itself can lead to protein breakdown, aggregation, and inactivation. And while there are many storage options, each has its own tradeoffs. Therefore, to help keep your proteins safe during storage, we’ll give you an overview of storage options along with six quick, handy tips!
1. Store in Solution at 4°C
Keep your concentrated protein in a simple solution (like PBS) in polypropylene tubes or clean glassware in the fridge.
- Pros: Your protein is easy to access and can be warmed quickly for assays. Additionally, you can resample from the same vial without worrying about free-thaw cycles that can speed up degradation.
- Cons: Microbial contamination, proteolysis, and oxidation are your enemies here, thanks to the higher temperature and environmental exposure when resampling. Avoid this by storing your protein in aliquots, adding protease inhibitors, antimicrobials and reducing agents (see our six handy tips below). Also, you should plan to toss your proteins out after less than a month when stored at this temperature.
2. Store in Solution With Cryoprotectant at -20°C
Preserve your protein in solution with 25 – 50% (w/v) glycerol or ethylene glycol in the freezer.
- Pros: Firstly, at this lower temperature, your proteins are more resistant to microbial contamination, protease degradation and oxidation. Diluting them with glycerol or ethylene glycol will keep them in solution, allowing you to resample easily. Furthermore, proteins stored this way can last for up to one year. This is a great overall option for antibody conjugates.
- Cons: Proteins in solution at -20°C may still suffer from proteolysis, contamination, and oxidation. Again, aliquoting, inhibitors, and reducing agents are your friends. Keep in mind that diluting your sample with up to 50 % cryoprotectant may not be desirable, depending on what you want to do next.
3. Freeze at -80°C or in Liquid Nitrogen
Snap-freeze your proteins in single-use aliquots and keep them frozen.
- Pros: Between the freezing temperature and lack of resampling, this is a great option for long-term storage (many years). In addition, you usually don’t need to dilute or contaminate your protein with chemicals (glycerol, sodium azide, etc.) to protect it from degradation and microbes.
- Cons: Unlike the previously mentioned solution-based options, frozen proteins should be viewed as single-use only in order to avoid degradation from repeated freeze-thaw cycles. Moreover, antibody conjugates (especially alkaline phosphatase conjugates) may not work after freezing.
Dehydrate your protein to a powder for indefinite storage in the freezer.
- Pros: Firstly, your protein will be protected from hydrolysis and other forms of chemical degradation that that can happen in water. Additionally, lyophilized protein takes up less room than solutions and is amenable to long-term storage (years).
- Cons: You need a lyophilizer (or a friend who has one!). Bear in mind however, that lyophilization may not be possible for proteins associated with cell membranes, which are readily disrupted by lyophilization. Similarly, not all proteins remain stable through lyophilization; they may denature, which reduces their usefulness and ability to be redissolved. Furthermore, some proteins may need to be stabilized with sucrose, mannitol, or BSA.
Six Handy Tips!
- Aliquot. Whether frozen or in solution, aliquot your protein into single-use tubes to avoid freeze-thawing and to reduce contamination risk.
- Concentrate. Dilute proteins are better able to bind to plastic or glass. Avoid this by either concentrating them to >1 mg/ml, or adding a “filler” such as BSA to reduce binding.
- Cryoprotect. Stabilize your proteins and prevent ice formation by adding 25 – 50% (v/v) glycerol or ethylene glycol.
- Inhibit microbial growth. Add thimerosal (0.01% w/v) or sodium azide (0.05% w/v) to reduce microbial growth.
- Inhibit proteases. Reduce protein degradation by adding protease inhibitors (e.g. EDTA, PMSF).
- Stop cysteine oxidation. Protect your –SH groups! Add 1 – 5 mM of a reducing agent (e.g. DTT, 2-mercaptoethanol).
We hope these recommendations keep your proteins safe and sound! To learn more about storing proteins, please check out the references below! Also, if you have any tips not mentioned here, do share them by writing in the comments section!
Deng J, Davies DR, Wisedchaisri G, Wu M, Hol WG, Mehlin C. An improved protocol for rapid freezing of protein samples for long-term storage. Acta Crystallogr D Biol Crystallogr. 2004; 60(1):203-4.
Matejtschuk P. Lyophilization of proteins. Methods Mol Biol. 2007; 368:59-72.
Protein stability and storage. Thermo Fisher Scientific, Inc. 2009.
Storage of purified proteins. Protein Expression and Purification Core Facility, European Molecular Biology Laboratory.