During protein purification, a protease inhibitor is added to the lysate (10ul/mL of lysis buffer). Why is this amount of inhibitor desirable? What is the potential danger to the protein if a greater volume of inhibitor is added?
I don't think there is much danger to the protein to adding more than the recommended amount, unless it is itself a protease. It just adds unnecessary cost.
Some protease inhibitor cocktails are supplied as a solution in DMSO solvent at 100X the final concentration, so that at 1X the DMSO concentration is 1%, which most proteins can handle. Adding more of such a cocktail risks causing damage to the protein if it is very sensitive to solvent.
I don't think there is much danger to the protein to adding more than the recommended amount, unless it is itself a protease. It just adds unnecessary cost.
Some protease inhibitor cocktails are supplied as a solution in DMSO solvent at 100X the final concentration, so that at 1X the DMSO concentration is 1%, which most proteins can handle. Adding more of such a cocktail risks causing damage to the protein if it is very sensitive to solvent.
If your protein is enzyme than these inhibitors can affect activity assay of enzyme. If your adding inhibitors with EDTA, they can chelate all metal ion which may be required for your protein stability or activity.
Hope this will help
Some protease inhibitors like PMSF covalently modify the active center of proteases e.g. PMSF react Serin residues. With increasing concentration the risk of unspecific modifications of Serins in your target protein might increase. This might destabilize your protein or destroy enzymatic or binding activity.
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