I know it could be a little naive question.
Wanted to gather views and experiences on likelihood of stock solutions of antibodies, in PBS (with sodium azide) likely to suffer degradation due to protease contamination, resulting from casual handling of the said antibody vials (outside a biological hood).
Has anyone suffered it and how can this be avoided?
You should make a distinction between purified antibodies and antisera. It seems to me that antisera would be more of a problem for storing at 4oC, since there could be proteases present in the sera. Purified antibodies should be essentially free from proteases.
Hello Rohan,
In my case, I experienced significant loss of antibody activity and degradation due to freeze-thaw issue. Secondly, solubility in buffer must be checked, if the protein is generated/ purified in house. For phosphate buffer system, pH 6.5 to 7.8 has been suggested to be optimal. You may like to check the literature on antibody aggregation in various buffers (Article added). Above all, it is prudent to work with aliquots of the antibody stocks and prefer freezing (-20 C or below) as storage temperature.
Hope you find it useful.
It is not necessary to handle antibody solutions in a biological hood. Keep the solutions on ice after they are thawed, handle them with normal care, and they will be fine.
Rohan Aggarwal Hi. I have had that happen. You should never store any important proteins like Abs at RT. Any trace proteinase will go to town if given the chance.
Since that debacle, I store all important proteins long-term at -80oC in one-time-use aliquots. At that temperature all hydrolases are mostly inactive because there is no free H2O around.
Many serine proteinases are still active at -20oC.
Thanks for your answers. We are storing our antibodies at 4 deg as per manufacturers guidelines. The protease shouldn't affect the antibodies at 4. Right?
You should make a distinction between purified antibodies and antisera. It seems to me that antisera would be more of a problem for storing at 4oC, since there could be proteases present in the sera. Purified antibodies should be essentially free from proteases.
Actually, with respect to proteinases, storing antibodies as serum is safer than storing them as purified proteins. Serum still has quite a bit of serine proteinase inhibitors (serpins) and other inhibitors in it.
https://www.jstage.jst.go.jp/article/tjem1920/139/3/139_3_265/_pdf
Most proteinases are perfectly happy at 4oC if there are no inhibitors around.
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