We recommend the following protocol for Azide-PEG3-maleimide generation with the Azido-PEG3-maleimide Kit:
- For the 25 mg kit, add 1 mL of a dry water-miscible solvent (like DMF or DMSO) to the azido-PEG-amine and shake for approximately 30 seconds to ensure mixing. For the 100 mg kit add 2.5 mL of solvent.
- Place the maleimide-NHS ester under nitrogen or dry atmosphere and slowly add the solution from step 1 with stirring or shaking.
- Stir or shake the reaction mixture for 30 minutes at room temperature. The progress of the reaction can be followed by Thin Layer Chromatograph.
Note: For TLC, the solvent is typically methanol:methylene chloride (1:20), run on a silica gel normal phase plate and developed with a potassium permanganate spray. The Rf of the azide-PEG3-maleimide is slightly lower than the Rf for the maleimide-NHS ester. When the reaction is complete, it will be one clean spot on the plate.
- The reaction product will be ready-to-use azide-PEG3-maleimide.
The azide-PEG3-maleimide is present at ~ 75 mM if 1 mL of solvent is used in step 1. The 25 mg kit produces 0.075 mmol of azide-PEG3-maleimide.