Alkylating Proteins or Peptides in Solution with NEM

1. Make the N-ethylmaleimide solution (NEM) fresh each day that you need to use it.  An easy way to prepare NEM in advance is to weight out correct amounts of it in eppendorf microfuge tubes, and store it in the dark at room temperature.  When using it, just add the appropriate amount of water.
2. Dissolve a stock solution of 100 mM NEM in nanopure water in an eppendorf microfuge tube.  Vortex if necessary.  Leave the solution on ice until ready to use.
3. Incubate the protein or peptide solution (which is in a buffer) in NEM at a final concentration of 5 mM to 50 mM NEM for 10 minutes on ice or at room temperature.
4. Dialyze away the NEM if desired.

Reducing Proteins or Peptides in Solution Prior to Alkylation

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.
3. 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.
4. Dialyze away the DTT or NEM if desired.

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