Hello!
I did some in vitro digestions according to the INFOGEST (Minekus) protocol. When I analysed by immunobloting the different aliquots of the digestion I observed "background" signals that correspond to the size of the various digestive enzymes: pepsin but specially intense are the signals from trypsin and chymotrypsin (these last ones of a similar size of my protein of interest).
I found some publications where is described that enzymes such trypsin and chymotrypsin can actually break up the substrate and produce chemiluminiscence...
Does someone know a way to avoid this?
Thank you in advance!
Hi Patricia,
Why don't you use specific inhibitors for these enzymes? You can also inactivate all these digestive enzymes by heat or another way, like different pH, salt concentration etc.
Good luck
thanks a lot for your answer! Actually I used Pefabloc to inactivate the enzimes, immediately after taking the aliquot (5 mM pefabloc in the aliquot)... that is why I didn´t understand those signals in the immunoblots... but they really match up with the digestive enzymes´sizes!
- Badges
- Science method