Protein storage

   When proteins are expressed and purified, many are unstable when not in their native environments such as specific cell compartments and extracellular fluids. If certain buffer conditions are not maintained, extracted proteins may not function properly or remain soluble. Proteins can lose structural integrity and activity as a result of proteolysis, aggregation and suboptimal buffer conditions.

   It’s important to remember depending the on nature of the protein and the storage conditions used. The shelf life of protein can vary from a few days to more than a year. The following are some general guidelines for protein storage and stability.

   There are tradeoffs associated with each method. For example, proteins at 4°C can be easily dispensed and avoiding freeze and thaw cycles but susceptible to microbial or proteolytic degradation. At 4°C, proteins may not be stable for more than a few days or weeks. Lyophilized protein can be stored for longer term without degradation, but the lyophilization process itself may damage the protein.


Storage conditions 4°C 25-50% glycerol or ethylene glycol at -20°C  Frozen at -20° to -80°C or in liquid nitrogen  Lyophilized and frozen  
Shelf life 1 month 1 year 1 year years
Antibacterial additive yes yes no no
number of use many many once once


Protein storage methods: 

  • Generally, proteins are best stored at ≤ 4°C in autoclaved glassware or polypropylene tubes. Storage at room temperature often leads to protein degradation and/or inactivity, commonly as a result of microbial growth. For short-term storage (1 day to a few weeks), many proteins can be stored in simple buffers (phosphate or Tris buffers) at 4°C. 
  • For longer storage, protein samples are dispensed and prepared in single aliquots then frozen at -20°C or -80°C to avoid repeated freeze and thaw cycle. Alternatively, by adding 50% glycerol or ethylene glycol to prevent protein solution from freezing at  -20°C is another way to avoid thawing process.
  • By dropping (100 ul) protein solution into a pool of liquid nitrogen to form frozen protein beads and store them in cryovials under liquid nitrogen. (Storage 1 month to 1 year)
Carrier proteins: 
  • When put into the storage vessel, small amount of proteins can bind to the surface. The low concentration solutions (< 1 mg/ml) are more prone to this effect. It is common to add certain carrier proteins such as bovine serum albumin (BSA) at 1-5 mg/ml to the diluted protein to prevent such degradation and loss.
Common additives: 
  • Cryoprotectants such as glycerol or ethylene glycol to a final concentration of 25-50% help to stabilize proteins by preventing the formation of ice crystals at -20°C that destroy protein structure. 
  • Protease and Phosphatase Inhibitor Cocktails provide broad-spectrum protection against proteolytic cleavage of proteins from trace proteases contaminant from the cell lysis and protein extraction.
  • Anti-microbial agents such as sodium azide (NaN3) at a final concentration of 0.02-0.05% (w/v) can inhibit microbial growth. 
  • Metal chelators such as EDTA at a final concentration of 1-5 mM avoid metal-induced oxidation of –SH groups and help maintain protein in reduced state.  
  • Reducing agents such a dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) at final concentrations of 1-5 mM can also be used to keep protein in reduced state.
Antibodies and Antibody-Enzyme Conjugates
  •  Antibody stock solutions (e.g., 1 mg/ml) in simple buffer can often be stored at 4°C for days to weeks without significant loss in activity. More than often, 50% glycerol or ethylene glycol may be added for increased stability at -20°C storage. In order to prevent repeated freeze-thaw cycles, antibody can be stored in small working aliquots at -20°C. This is especially important to antibody-enzyme conjugates such as alkaline phosphatase conjugates.  Anti-microbial agents such as sodium azide or thimerosal often are added to avoid microbial growth. Ethylene is a better choice in terms preventing microbial growth.