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Proteins can be unstable out of their native environment. They can lose activity as a result of proteolysis, aggregation, and suboptimal storage. Optimal conditions for storage depend on the specific protein, and instructions can be found on the datasheet. Please find some general guidelines below.
Please find some general guidelines for protein stability below.
Lyophilization allows for more stable long-term storage; however, the protein must be reconstituted before use. There are also specific considerations that need to be taken for handling lyophilized peptides and proteins.
Protein storage in solution is more prone to microbial or proteolytic degradation.
Storage of protein solutions at room temperature leads to protein degradation and/or inactivity, also commonly as a result of microbial growth.
Repeated freeze-thaw cycles should be avoided, which can also decrease protein stability.
Reconstitution concentration is important for protein stability.
Solutions at concentrations lower than 1 mg/mL are more prone to proteolysis or inactivation because of low-level binding to the storage vessel.
Because it may not be realistic in some cases to have a higher concentration than 1 mg/mL, it is common practice to add “carrier” , such as purified bovine serum albumin (BSA), to 1 to 5 mg/mL (0.1% to 0.5%).
Some of the additives below may help to enhance the shelf life of the proteins. However, the question of whether to add or not should be carefully considered depending upon the experimental need.
For example, high viscosity may compromise sample quality as well as downstream experimental results. While helping to prevent disulfide bond formation, reducing agents also inhibit other redox reactions which may be of interest. Similarly, the inclusion of protease inhibitors for proteins to be used with cell cultures or in living systems would not be advisable.
Adding 25-50% glycerol or ethylene glycol can help to stabilize proteins by preventing the formation of ice crystals at -20 °C, that can destroy protein structure, enabling repeated use from a single stock.
Protease inhibitors interact with the active site of proteases that hamper proteolytic activity detrimental to protein stock stability. Some examples are: PMSF (0.1-1 mM), Pepstatin A (1 μg/mL), Leupeptin (1 μg/mL), etc.
Phosphatase inhibitors inhibit several phosphatases to preserve the phosphorylation state of the proteins.
If you don't add phophatase inhibitors, this can result in reduced or no kinase activity or unexpected protein size.
Adding antimicrobial agents—for example, 1.02-0.05% (w/v) sodium azide (NaN3) or 0.01% (w/v) thimerosal—can help to prevent microbial growth.
Most proteins contain free thiol groups containing cysteines and oxidation of these free thiols leads to the formation of a disulfide bond between cysteines. The majority of the intracellular proteins require these free thiols for their biological function.
Adding reducing 1-5mM dithiothreitol (DTT), and 2-mercaptoethanol (2-ME, sometimes called ß-ME), can help to maintain proteins in a reduced state by preventing oxidation of cysteines.
Please note that the presence of reducing agents can interfere with certain reactions. So we recommend removing reducing agents if you intend to conjugate the protein. Since reducing agents prevent disulfide bond formation, this is particularly pertinent if you intend to label the protein via a thiol group. In addition, some protein structures are maintained by intramolecular disulfide bonds, which might be broken in a reducing environment.
Adding chelating agents, like 1-5mM EDTA, groups proteins together, and helps to maintain the reduced state.
Adding detergents such as 0.01% Tween-80 help maintain the stability of the hydrophobic protein in aqueous solution. This also reduces the probability of proteins binding to the storage tube.