Note: PTAD-Alkyne must be first activated by stirring in a 1:0.98 molar ratio with 1,3-dibromo-5,5-dimethylhydantoin (product #157902). Activation is evident upon solution color change from colorless to deep red and the activated reagent should be used immediately.General Procedure for Protein Modification with PTAD.Part 1: PTAD activationMix together 1:0.98 molar equivalents of unactivated PTAD to 1,3-dibromo-5,5-dimethylhydantoin (product #157902) in organic solvent (preferred solvents are DMF or acetonitrile; avoid using DMSO)Color change is observed from colorless/pale yellow to deep red (approximately 5 min of mixing).After the solution turns red, store the now activated reagent on ice and use for protein modification within 30 min.Part 2: Protein modificationAdd protein solution in mixed phosphate/Tris buffer or Tris buffer (pH should be 6 - 9) to the eppendorf tube (or other vial) containing the activated PTAD reagent prepared above and mix gently at room temperature for up to 30 min. Preferably use 10-fold molar excess of reagent relative to protein. Use protein at a minimum concentration of 1 mg/ml (higher concentrations are preferred for enhanced labeling).Remove excess unreacted PTAD by gel filtration.
