Hello everyone,
I am trying to perform a co-IP pf a protease. As you know, some protease inhibitors will be added to lysis buffer during IP sample preparation. So if the interaction between this protease and its binders will be affected by those protease inhibitors which may produce false negative results? If yes, what can I do to avoid it ?
Best,
Jing
One of the ways is isolation of organelles. If your protease is in cytoplasm there would be no need to save it from lysosomal proteases, for example, so protease inhibitor cocktail would not be needed.
But I'm not sure about how to do it. Maybe it won't help.
Protease inhibitors work by binding to or reacting with the active site of the protease. So, if the interaction site of the protein-protein interaction is outside the active site, the protease inhibitors should not affect it.
Hi Adam B Shapiro,
so in your opinion, I should do some analysis on protein-protein interaction mannually and confirm those interaction sites? If yes, could you please recommend me some useful tools? Your help is greatly appreciated.
Thank you.
Best,
Jing
Sorry, I don't know how to do this. Perhaps someone else can make a suggestion.
To investigate if your protease interacts with the candidate protein through its active site I suggest that: Just perform co-ip with protease inhibitor. If there is no interaction then, probably, the interaction goes through the active site. Then try to do the same without inhibitor and theoretically the interaction will occur.
Great thank to Adam B Shapiro and Valentina Bozok. Someone just told me a ignored truth that the corelated proteins have formed this interaction before processing, although protein inhibitors may inhibit their interaction, they can't break the existed interaction. I think it makes sense.
Best,
Jing.
That depends upon the affinity of the protein-protein interaction. If it is strong enough, then there will be very little of the unbound form of the protease present at equilibrium to react with the inhibitor. But if the interaction is less strong, there may still be competition.
Dear
We can note that once interaction between protease and its binders has done. Their are not site available for inhibitors to react. You have in your experiment to avoid contact of protein with inhibitors as lysosomal proteases. So inhibitors could attach to protein with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. And yet the solution to resolve inhibitors problem is to remove its by dilution or dialysis.
Some references could help.
-Cleland WW (1963). "The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory". Biochim. Biophys. Acta. 67: 173–87.
Hope to be helpful
Marius
https://www.ncbi.nlm.nih.gov/pubmed/14021668
Thank you for your great advice and suggestion.
Have a nice day.
Best
Jing
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