Can anyone address me to any protocol describing the best way to store my proteins (proteases) in glycerol and how to remove it before usage?
If diluting the enzyme is not enough (compare the use of restriction enzymes), a quick gel filtration (buffer exchange) will do the trick. Depending on the volume you need to treat, PD-10, NAP-10 or smaller columns are available (look for Sephadex G-25 or similar as resin) or make your own.
You also could try snap-freezing aliquots in PBS in liquid nitrogen and storing them at -80°C, or try lyophilisation.
If there is no reference (established protocol) for your enzymes, I'd strongly suggest not to trust what you can find for other proteins, and thoroughly test (time course), if it works for your proteins to preserve their activity.
To store proteins unfrozen at -20oC, the storage buffer should contain 50% glycerol by volume. For storage frozen at -70 or -80oC, 10-20% is common.
The stock solutions of proteins are usually very concentrated compared to the concentration used in enzyme reactions, so replacement of the storage buffer is not necessary because it is diluted so much. For applications in which the protein concentration is similar to the stock solution concentration, the buffer can be replaced by dialysis (which is a slow method), or gel filtration (such as with a spin column, which is a fast method, but with some loss of protein, as described by Wolfgang Schechinger above), or by using a centrifugal ultrafiltration device to concentrate and redilute.
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