Page 128 - Tyrosine-Based Bioconjugations - Jorick Bruins

Appendix
Materials
Prepare stock solutions of buffers in 18 MΩ·cm water (MilliQ). Protein stock solutions can be stored for short-term usage at 4 °C, or for long-term storage at –80 °C. Specific cpTCO-probes and BCN-probes should be stored long-term at –20 °C in dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF) or N,N-dimethylacetamide (DMA). BCN containing an alcohol functionality (BCN-OH; CAS 1263166-90-0) can be synthesized4 or purchased from commercial vendors. Functionalities can be introduced via standard organic chemistry methods. cpTCO containing an alcohol functionality (cpTCO-OH) can be synthesized5, 11 and functionalities can also be added via standard organic chemistry methods. Mushroom tyrosinase can be purchased from several vendors (CAS Number 9002-10-2).
Materials - SPOCQ reaction
1. Phosphate Buffered Saline (PBS) buffer: 50 mM sodium phosphate,150 mM NaCl, pH 5.5 (see Notes 1,2).
2. Phosphate buffer: 50 mM sodium phosphate, pH 6 (see Note 2).
3. Protein solution: Protein of interest constituted in PBS buffer (see Notes 3) (e.g.
trastuzumab containing a G4Y-tag on its heavy chain; Tras[HC]G4Y).
4. Stock solution of cpTCO-probe: ≥0.4 mM solution of any cpTCO probe (with attached
fluorophore, toxin or other molecule; e.g. cpTCO-Lissamine) in DMSO (see Notes 4–6).
5. Stock solution of BCN-probe: ≥0.4 mM solution of any BCN probe (with attached fluorophore, toxin or other functional group; e.g. BCN-Lissamine) in DMSO (see Notes 4–
6).
6. Mushroom tyrosinase solution: 0.5–10 mg/mL of mushroom tyrosinase in 50 mM
phosphate buffer, pH 6 (see Note 7,8).
7. A tabletop thermoshaker for controlling the reaction temperature (e.g. Eppendorf
ThermoStatTM C).
Materials - Purification
1. Amicon® ultra-spin filtration units with the appropriate molecular weight cut-off (MWCO) for the selected protein (see Note 9).
2. Phosphate Buffered Saline (PBS) buffer: 50 mM sodium phosphate, 150 mM NaCl, pH 7.2
3. For further purification, depending on the protein of interest, affinity based columns can be used, e.g. Protein A columns for antibodies, Nickel-NTA columns for proteins with a
histidine tag.
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