A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules

The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules such as proteins and peptides. Maleimides are electrophilic compounds which show high selectivity towards thiols.
1. Dissolve the peptide or other biomolecules containing thiol in degassed buffer (PBS, Tris, or HEPES) at pH 7-7.5.
2. Add a 100x molar excess of TCEP (tris-carboxyethyl phosphine) reagent to reduce disulfide bonds.
3. Dissolve maleimide in DMSO or fresh DMF (1-10mg in 100uL).
4. Add dye solution such as cy5 maleimide to thiol solution (20x fold excess of dye), flush with an inert gas, and close tightly.
5. Mix thoroughly and keep at room temperature or 4C overnight.
6. Purify by gel filtration, HPLC, FPLC, or electrophoresis.