i am currently transfecting a plasmid that express a protease, and this protease predicted to cleavage my target protein X by bio information prediction. In order to see my target protein X cleavage product, i used a C-terminal specific recognition protein X antibody, but still did not saw any cleavage product, but compare to the control (with not transfect protease expressing plasmid, the total amount of protein X reduce.
Hi,
Are you sure that your protease is expressed and active in your cells ?
Is your loading control the same for the protease-expressing cells and the non-expressing cell ?
What is the expected size of the cleave product ?
thanks, the protease were well expressed in cells and my loading control were the same between both cells. i have tried MG132 , proteosome inhibitor but it doesn't work.
Purifiy protease and Protein X, then perform your assay in vitro to have a proof and not bioinformatic prediction. Correct me if I wrong, but you verify protein cleavage in your expression system, and after the cleavage of your protein X the remaining parts could be very easily degraded.
You can detect protein cleavage by several methods:
1. by inhibition experiments (comparison of SDS-Pattern +/- protease inhibitors like PMSF, depending on the protease type you are expecting)
2. by initiation: adding specific proteases into your sample. Compare +/-
3. by Western blotting with a monoclonal Ab for your protein. Just have a look on the potential cleavage sites to get some ideas about the size (mol. weight) of the expected fragments). If the epitope of the Mab is only once present (and not within the cleavage site), the uncleaved protein should be larger the the cleaved one. Therefore it could be helpful, to use a set of Mab's recognizing epitopes on different fragments. Inhibition of proteases and adding of proteases is helpful for the interpretation of the results.
4. Western blotting using polyclonal antibodies. They can recognize (probably) epitopes on all fragments.
But: BEWARE OF CROSSREACTIONS OR UNSPECIFIC REACTIONS ... and protease inhibitors can not used in cell cultures, because they are often toxic.
If you know the amino acid sequence of the suspected cleavage products, you could target them by mass spec analysis using Selected Reaction Monitoring to identify them and then measure their abundance in your cell lysate. However, this is a specialized technique that requires you work with someone with expertise in this area...and it is a bit pricey...
It seems that you protein X is digested in multiple sites by the protease, so you can't detect the expected c-terminal fragment of your protein X by SDS-PAGE because it generates too small fragments (peptides). In order to confirm this hypothesis you could analyze the resulting peptides after digestion by MALDI-ToF MS.
Anyway decreasing the amount of protease, and/or the digestion time and/or the temperature could reduce this putative aspecific effect.
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