One should add aprotinin before centrifugation of samples for plasma or serum isolation and then freeze for subsequent small peptides' determination. If this step has been missed, is there any sense in adding aprotinin after thawing such samples?
Hi Chrysanthi, plasma and serum is loaded with proteinase inhibitors.
Then again, plasma and serum is also loaded with proteinases in their zymogen form or active proteinases in complex with inhibitors.
I would err on the side of caution and add aprotinin, because many serine proteinases are active at -20oC.
Dear Chrysanthi, although as you say the addition of the antifibrinolytic agent like aprotinin should be done rather before freezing of plasma, my feeling is that it would be a good strategy to add it at the time of thawing of plasma at +/- 30-35°C (as soon as the plasma is completely thawed) in order to prevent proteolytic degradation during further processing steps. If the blood sample has been collected properly with good mixing with the anticoagulant, kept at 20-24°C to limit the risk of platelet activation, and centrifuged quickly to separate plasma from the blood cells, proteolytic activity would be maintained at low level before plasma freezing. Kind regards, Thierry
Dear Steingrimur, thank you very much for your answer. If many serine proteases are active at -20C, then what is the sense of adding aprotinin once thawed? I am not sure I understand this point
Hi Chrysanthi, sorry, my mistake. Thierry has the right answer in that aprotinin should be added before freezing.
Hi Steingrimur, thanks for your additional comment. I agree with you on the fact that plasma contains a balance of proteases and anti-proteases. As a matter of precaution, aprotinin could be added before freezing (ideal) and, if not, right after thawing to limit the risks of protease activation that results from thawing. The choice of the anti-proteases can include aprotinin, that inhibits fibrinolysis and the intrinsic coagulation pathway, or others. Benzamidine has been used at lab scale for its anti-protease activity. Best, Thierry
Hello Chrysanthi Skevaki ,
Sorry to revive this question with an other question.
At what concentration should we add aprotinin to serum to prevent inhibit proteases?
Sigma sells it at 10mg/ml (5-10 TIU/ml) but how much should be added to serum preparation?
Thank you for your help!
Karine
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