General Protocol for Reducing cysteines in proteins or peptides Solutions
- Make diothreitol DTT 1M stock solution in water, it is best to make fresh. (Try not to inhale DTT.)
- Add DTT to the protein or peptide solution (the buffer) to a final concentration of 1 mM to 10 mM DTT. Incubation for 10 minutes. 30 minutes. reduction can work a little better if incubating at a higher temperature, such as at 37 degrees C or 56 degrees C. However, room temperature or ice should work as well.
It may be necessary to change the nature of the protein or peptide first first before DTT reduction. For example, it is possible to use urea (1-8 M concentration) or SDS (0.1 to 4%) to first expose the cysteine for a few minutes on the ice or at higher temperatures. Alternatively, it is possible to alter the properties and reduce at the same time.
Reducing protein or peptide in Solution Before Alkylation
- Reduce protein as above.
- Incubation of the disulfide-reduced protein or peptide samples on NEM molarity of at least 3-fold higher than that used for DTT. Incubation for 30 minutes. for 1 hour.
- Dialyze eliminate DTT or NEM if desired.