Reducing Proteins or Peptides with Dithiothreitol (DTT)
Biochemistry, Molecular Biology, and Cell Biology Protocols >> Reducing Proteins or Peptides with Dithiothreitol (DTT)
General Protocol to Reducing the Cysteines in a Protein or Peptide Solution
1. Make a 1M diothreitol DTT stock solution in water, best to make fresh. (Try not to inhale the DTT.)
2. Add DTT to the protein or peptide solution (which is in a buffer) to a final concentration of 1mM to 10 mM DTT. Incubate for 10 min. to 30 min. The reduction may work slightly better if incubating at higher temperature, such as at 37 degrees C or at 56 degrees C. However, room temperature or ice should work too.
It may be necessary to first denature the protein or peptide first prior to DTT reduction. For example, it is possible to use urea (1-8 M concentration) or SDS (0.1-4%) to first expose the cysteines for a few minutes on ice or at higher temperature. Alternatively, it may be possible to denature and reduce at the same time.
Reducing Proteins or Peptides in Solution Prior to Alkylation
1. Reduce the protein as above.
2. Incubate the disulfide-reduced protein or peptide samples at an NEM molarity of at least 3-fold higher than that used for DTT. Incubate for 30 min. to 1 h.
3. Dialyze away the DTT or NEM if desired.