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Bench Tips:
Keeping Your Cool When Storing Purified Protein

Abstract

The study of proteins and their function is central to understanding biology, but separating and purifying single proteins from complex mixtures is hard work. After investing the time to purify your target protein using various chromatography methods, losing your protein integrity because of improper storage would be devastating. Good storage conditions are particularly important if you need your protein for several downstream studies. You might need to store your preparation for an extended period of time, and some experiments require that the protein retain its original structure, binding affinity or enzymatic activity. Optimal conditions for storage are distinctive to each protein and situation; nevertheless, it is possible to suggest some general guidelines for protein storage and stability. Below are some quick and simple suggestions relating to concentration, additives, sample size and storage temperature to consider when storing your protein for future use.

Concentration

Concentration plays a key role in keeping proteins stable in solution. Proteins need to be stored at a high level of concentration—at least 1 mg/ml. Dilute protein solutions (< 1 mg/ml) are more prone to inactivation and loss as a result of low-level binding to the storage tube. Because it may not be realistic in some cases to have a higher concentration, it is common practice to add “carrier” or “filler” protein, such as purified bovine serum albumin (BSA), to 1 to 5 mg/ml (0.1% to 0.5%), to dilute protein solutions and protect against such degradation and loss.

Additives

Many compounds may be added to protein solutions to lengthen shelf life. What you add to your protein solution depends on the intended use and the characteristics of your purified protein. For example, if you purified an antibody protein to be used in assays such as Western blotting or immunoprecipitation experiments, you could add cryoprotectants such as glycerol or ethylene glycol to a final concentration of 25% to 50% to help stabilize proteins. These additives help keep solutions from solidifying upon freezing (-20°C) by preventing the formation of ice crystals that destroy protein structure. Polymers like polyethylene glycol (PEG) and polysorbates such as Tween 80 can also help with keeping ice crystals at bay, but at much lower concentration (0.01%). Because your protein preparation will not freeze, it can be used many times without a need for thawing. In addition, at this concentration, Tween 80 can also help keep your protein from binding to your storage tube. However, cryoprotectant impurities may affect protein activity, so you might want to take steps to ensure the quality of the chosen reagent by checking with the vendor. Another consideration is that although these additives are good for antibody use, the high viscosity can compromise protein use in certain downstream procedures, such as additional chromatography steps, protein binding/interaction assays or mass spectrometry analysis, so other applications should be considered.

Protease inhibitors can be added to prevent proteolytic cleavage of proteins, thus keeping the protein intact. Antimicrobial agents such as sodium azide (NaN3) at a final concentration of 0.02% to 0.05% (w/v), or thimerosal at a final concentration of 0.01 % (w/v), inhibit microbial growth. Metal chelators such as EDTA at a final concentration of 1 to 5 mM avoid metal-induced oxidation of sulfhydryl (–SH) groups and help to maintain the protein in a reduced state. Reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) at final concentrations of 1 to 5 mM also help to maintain the protein in the reduced state by preventing oxidation of cysteines. Also keep in mind when mixing your additive with your protein that protein solutions should not be vigorously shaken by vortexing. It not only generates bubbles and raises oxygen levels in the solution, but can also affect protein structure because of the high shearing forces.

As you can see, there are many additive choices. Vendors such as Bio-Rad offer reagents in convenient sizes and have protocols and tips for which additives to use. Be sure to read and plan your downstream experiments to ensure the proper storage conditions.

Sample size

If you determine that using either glycerol or ethylene glycol is not good for your application, and because freeze-thaw cycles decrease protein stability, you should dispense and prepare single-use aliquots so that, once thawed, the protein solution will not have to be refrozen. These small aliquots can be used one time. The trick is to plan ahead and avoid multiple freeze-thaw cycles.

Temperature

Generally, proteins are best stored at ≤ 4°C in clean, sterilized glass containers or polypropylene tubes. Storage at room temperature often leads to protein degradation and/or inactivity. For short-term storage of one day to a couple weeks, many proteins may be stored in simple buffers containing protease inhibitors at 4°C. For mid-term storage, one month to a year, freezing at -20°C or -80°C is the more common form of cold protein storage. For serious long-term storage of up to one year, some researchers choose to bead single-use aliquots of the protein in liquid nitrogen for storage in clean plastic containers under liquid nitrogen. This method involves adding the protein solution dropwise (approximately 100 µl/drop) into a pool of liquid nitrogen, then collecting the drop-sized frozen beads and storing them in cryovials under liquid nitrogen. Depending on your situation and the properties of your protein, you might favor one storage temperature and storage duration over another. Be sure to keep in mind the intended applications, as well.

Following these simple recommendations will help to keep your protein stable and useful for your next experiments. Consider using the websites of vendors such as Bio-Rad for guidance in selecting the latest equipment and reagents to make protein purification easy and for insightful tips on storing your protein solution. Vendors may even be able to provide custom solutions tailored to your protein of interest. Finally, look for a company with strong customer-service programs. Talking to customer service for storage advice might just save your important protein samples as well as your precious time!