When you see two or more real bands for a protein what software do you suggest most useful for predicting the possible intracellular protease cleavage sites?
I've had good luck with http://web.expasy.org/peptide_cutter/. Seems to work well for actually predicting what the experimental cut sites will be.
It seems that you're just looking for a general prediction website for all proteases found in the intracellular environment. If that's the case, I've never come across something like that.
Hi Asim, why are you not using a proteinase inhibitor cocktail for the sample?
I did use a protease inhibitor cocktail for the sample during preparation of protein extract. Actually, the protein of our interest is cleaved at lease at three specific sites intracellularly. That's why I am looking for a tool for predicting protease cleavage sites.
Interesting. Intracellular proteinase activities are usually very specific for secreted proteins. Are you looking at pre-pro proteinase cleavage of a secreted protein? They should have specific signal sequences.
If this is not a secreted protein and the cells are healthy, then the proteasomes are the most likely culprits for cleaving intracellular proteins through ubiquitin modification.
If the cells are dying through either apoptosis or necrosis, then all hell breaks loose. You have lysosomal proteinases, caspases running rampant through the cells and chewing everything in site.
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