Alkylating proteins or peptides in Solution with NEM
- Make a solution of N-ethylmaleimide (NEM) is fresh every day you need to use it. An easy way to prepare in advance for the heavy NEM out the correct amount in eppendorf microfuge tube, and store it in the dark at room temperature. When using it, just add the right amount of water.
- Dissolve 100 mM stock solution of NEM in nanopure water in Eppendorf microfuge tube. Vortex if necessary. Leave the solution on ice until ready to use.
- Incubation of protein or peptide solution (the buffer) in NEM at a final concentration of 5 mM to 50 mM NEM for 10 min on ice or at room temperature.
- Dialyze go NEM if desired.
Reducing protein or peptide in Solution Before Alkylation
- 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.
- 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.