I am attaching a protein to a protease-substrate-PEG conjugate (nanogel). I encapsulated them within a hydrogel with a protease to measure the kinetic release of the protein. I wonder about the best techniques used to verify the cleaving of the protein by the protease from the conjugate?
The solution lies in the way which can detect the change in structure (bond or functional group disappearance)
Cleavage of protein May be detected through Raman spectra or by FTIR spectra of dried specimens before and after the reaction...
You can also proceed for protein assay with and without protease.
Wishes,
Hi Safaa, can you please describe your system in more detail?
Are you attaching your protein to a chromogenic proteinase substrate or to a peptide that is a target for proteinases?
If you have no chromogenic/fluorogenic changes upon proteinase cleavage, then you cannot easily follow the reaction in real time.
Probably the best way to follow the proteinase cleavage then is to run SDS-PAGE of the reaction with time and quantify the products.
Many thnx Steingrimur Stefansson for your answer! I am attaching my protein to a peptide that is a substrate for proteinases!
Kindly, if you have a protocol to run SDS-PAGE with time and quantify the products for short peptide, i will be grateful!
Hi Safaa, does your assay only have purified proteins in it or is it an assay that has many other proteins?
If it is an assay with purified proteins, then you can run it on regular SDS-PAGE. You can use SDS-PAGE gradient gels depending on the size of your product and just do a Coomassie or silver stain of gels. (Protocols for choosing gradient gels for optimal peptide separation and peptide staining are on the web)
Your controls on the gel should be your intact conjugate and in a separate lane, your products.
Your sampling time depends on how fast your proteinase is. Try first the extremes: 0 hr and 24 hr. Then narrow it down.
If you have an impure system, then you would have to do western blots if you have an antibody against your product and follow the same protocol.
Many thanks Steingrimur Stefansson for you explanation. My system has pure proteins, besides the peptides...
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