Page 130 - Tyrosine-Based Bioconjugations - Jorick Bruins

Appendix
2. Add 1 volume equivalent of 0.4-4.0 mM label (e.g. BCN covalently attached to a polyethylene glycol (PEG) group (BCN-PEG)); cpTCO covalently attached to a lissamine group (cpTCO-lissamine) in DMSO (resulting in 3-10 equivalents of label compared to G4Y) and mix properly by pipetting up and down 3 times. (See note 3).
3. Finally, add 1 volume equivalent of 0.5-10 mg/mL mushroom tyrosinase (resulting in 2.5-10 mol % mushroom tyrosinase) and mix properly by pipetting up and down 3 times (See notes 11, 12).
4. After the appropriate reaction time (0.5-5 hour), the reaction is available for purification, workup (e.g. Dithiothreitol (DTT) denaturation) and/or analysis (e.g. HPLC, LC-MS) (See note 13).
Example: 100 μL reaction of labelling Trastuzumab containing a G4Y-tag on its heavy chain (Tras[HC]G4Y) with fluorophore cpTCO-lissamine3
1. Add 80 μL of 10 mg/mL (67 μM) Tras[HC]G4Y solution (in PBS, pH 5.5) to a 1.5 mL Eppendorf tube and cool to 4 °C in a thermostat machine.
2. Add 10 μL of a 5 mg/mL (5.8 mM) cpTCO-lissamine stock solution to the Tras[HC]G4Y solution and mix the sample by pipetting up and down 3 times.
3. Add 10 uL of the 10 mg/mL mushroom tyrosinase solution to the mixture and mix the sample by pipetting up and down 3 times.
4. React the sample for 1.5 h at 4 °C.
5. Purify the sample according to section 5.2 and analyse using SDS-PAGE (section 5.3) or
HPLC (section 5.4).
In this example, the Tras[HC]G4Y had a final concentration of 8 mg/mL (53 μM), the mushroom tyrosinase a concentration of 1.0 mg/mL (1000 units/mL), and the label a final concentration of 0.5 mg/mL (580 μM). This would equal 10.9 equivalents of label, but because the symmetrical antibody has two G4Y tags, the excess label is halved to 5.5 equivalents. This method is generally applicable for any protein with the aforementioned tyrosine tag.
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