If the target protein is sensitive to DTT, are there alternative means to induce on-column cleavage?
If the activity of the target protein is affected by high concentrations of DTT or 2-mercaptoethanol, lower concentrations of DTT or 2-mercaptoethanol (5-10 mM) may be used for on-column cleavage. However, longer incubation time or higher temperatures (up to room temperature) may be required for efficient cleavage. Alternatively, 50 mM of freshly prepared hydroxylamine (for pTYB1 and pTYB2) or cysteine solution (at pH 8-9) can be used to induce cleavage at 4-25°C. Be aware that when hydroxylamine or cysteine is used with pTXB1,3, pTYB1,2,3 or 4, they form a stable covalent bond with the C-terminus of the target protein. One should determine whether a C-terminal hydroxylmate or cysteine affects the activity of the target protein. When cysteine is used for cleavage with pTYB21, pTYB22, pTYB11 or pTYB12, the cysteine is not attached to the target protein. If hydroxylamine or cystein is used the C-terminal thioester is not generated. If the recombinant protein is to be used in a ligation r
Related Questions
- Are there known ways to accelerate the hydrolysis of DTT from the C-terminal of the target protein in a controlled manner?
- How can an on-column cleavage reaction be carried out if the target protein has optimal activity at low salt?
- If the target protein is sensitive to DTT, are there alternative means to induce on-column cleavage?
