Worthington Biochemical FAQ

What are some guidelines for clostripain usage?

Clostripain cleaves peptides at the C-terminal side of arginine residues, calcium is required for maximium activity. Reducing agents like DTT, cysteine or mercaptoethanol are considered activators as they protect from inactivation by oxidation. The enzyme is inhibited by oxidizing agents, sulfhydryl reactants, and heavy metal ions. Citrate, borate and Tris anions are less inhibitory.

Optimium conditions for use will have to be determined empirically but I have tried to answer general questions below.

1. what protein concentration should I use?
        typical ranges of enzyme to sample protein are 1/20 to 1/100

2. How long will it take?
        Incubation time is dependent on nature of the the protein to be digested and temperature- typical range- 1 to several hours at 37°C.

3. What buffer to use?
        Buffer of choice containing calcium 1mM and 2.5mM DTT (or mercaptoethanol) pH range 7.4-7.8

Can initially reconstitute clostripain in water at 1 mg/ml aliquot and freeze(avoid repeated freeze thaw cycles)

The following references/link are using clostripain for protein cleavage and may help you develop a protocol: