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Protect proteins with cryoprotectants & protein concentration

Posted by The Protein Man on Aug 1, 2017 2:30:00 PM

Protein StructureRough handling and proteins do not necessarily go well together. As proteins are extremely sensitive and can easily be degraded during the process of concentration, purification, and storage, they need to be handled with utmost care to reduce, if not eliminate, the risk of unwanted aggregation and/or degradation.

Additionally, careful handling can also preserve the integrity of your precious protein samples during storage. This is very important since you may need to retain their structure, binding affinity, and enzymatic activity if you intend to use them for several downstream studies.

So, what should you do to safeguard the integrity of your protein samples during storage? While there are no hard and fast rules that apply to all proteins (each protein requires different storage conditions for optimal results), here are some general guidelines that can help keep your proteins safe during storage.

Storing Concentrated Protein Samples:
How to Do It Right

Concentrate your protein samples. Since diluted protein samples are more susceptible to binding to the storage tube, you should always store them at higher levels of concentration. For best results, try to concentrate them to at least 1mg/ml. If this is not possible, add a carrier or filler protein (e.g. BSA) to reduce the risk of unwanted binding and degradation.

Store at low temperatures. Storing proteins at room temperature is a bad idea since it increases the risk of degradation and loss of activity. To avoid getting yourself and your precious proteins into trouble, store concentrated protein samples in clean, sterilized glassware or polypropylene tubes at 4oC. If you will be using them within two weeks, you can just store them in simple buffers containing the appropriate protease inhibitors, antimicrobials and reducing agents. Storing your proteins under these conditions will make them readily available for use. Plus, you can easily resample from the same vial without subjecting your samples to freeze-thaw cycles that can hasten the degradation process.

However, if you don’t plan on using them in the immediate future and would like to store them for one month or even up to a year, consider freezing concentrated protein samples in solution with an appropriate cryoprotectant (e.g. 25-50% (v/v) glycerol or ethylene glycol) at -20oC. Storing your protein samples under these conditions reduces the risk of oxidation, protease degradation and microbial contamination, prevent damaging ice formation and makes resampling a breeze. 

For long term storage, snap freezing your proteins in single aliquots at -80oC may be your best choice. Aside from keeping your valuable proteins viable for many years, there is no need to contaminate or dilute them with chemicals.

Use appropriate additives. You can use a number of additives to prolong the shelf life and protect the integrity of your precious protein sample. However, these will depend on the nature of your purified protein and its intended application. Unless you are subjecting your sample to additional chromatography steps or mass spectrometry analysis, you can use cryoprotectants to stabilize your proteins and prevent the formation of ice crystals upon freezing.

You can also use protease inhibitors (to prevent proteolytic cleavage of proteins), antimicrobial agents such as sodium azide or thimerosal (to inhibit microbial growth) and metal chelators such as EDTA or reducing agents such as DTT or 2-ME (to keep the protein in a reduced state).

Prepare single-use aliquots. Storing concentrated protein samples in single-use aliquots eliminates the need for additives that may compromise the integrity and quality of your precious protein samples. It also minimizes the risk of contamination and allows you to do away with having to thaw your samples prior to use which may negatively affect the stability of your protein sample.

By following these suggestions, you can keep your protein samples safe, even if you plan on storing them for years.

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Topics: Protein Concentration

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