Page 132 - Tyrosine-Based Bioconjugations - Jorick Bruins

Appendix
Size-exclusion is most easily performed using Amicon® ultra-spin filtration units with the appropriate MWCO (see Note 9). This will remove any residual label and allows for concentration of the sample and/or buffer exchange.
1. Pre-wash the ultra-spin filtration unit by adding 500 μL of the buffer of choice (e.g. PBS pH 7.2) and centrifuge at 14.000 x g to a final volume of 25-50 μL.
2. Pipet the sample into a pre-washed 0.5 mL ultra-spin filtration unit and dilute to 500 μL.
3. Centrifuge at 14.000 x g to a final volume of 25-50 μL. The time depends on the MWCO. Please consult the specifications of the selected spin filtration unit.
4. Wash and exchange buffer to the buffer of choice by diluting to 500 μL with the buffer of choice and concentrate by centrifuging identical to step 2. Repeat this step twice to a total of three times and concentrate by centrifugation until the desired concentration is achieved. Determine concentration via UV/Vis spectrophotometry.
5. Further purification can be performed, e.g. to remove mushroom tyrosinase via a nickel-NTA column (if a histidine-tag is available), or in the case of an antibody, protein A affinity chromatography. For small volumes, ThermoFisher Scientific NAbTM Protein A Plus Spin Columns and comparable products are suitable.
Methods - SDS-PAGE
General SDS-PAGE workup for SPOCQ performed on antibodies (See note 14):
1. Prepare acrylamide gel according to standard protocol from Bio-Rad or other source.
2. Dilute 5 μg of antibody solution 1:1 with SDS-PAGE sample buffer SB (2×) including 5% BME.
3. Heat for 5 minutes at 95 °C.
4. Apply the denatured sample for SDS-PAGE analysis (12% acrylamide gel; with Bio-Rad dual
colour standard is an advised reference protein ladder).
5. If a fluorescent probe is added, measure fluorescence with the required equipment (e.g.
BioRad ChemiDocTM systems).
6. Stain SDS-PAGE gel with staining solution for 30 minutes.
7. Destain SDS-PAGE gel with destaining solution for 60 minutes.
8. Re-equilibrate SDS-PAGE gel in demiwater for at least 2 hours. Analyse gel with a scanner
or gel documentation system.
Example: After performing the SPOCQ reaction according to the procedure described in section 3.1, SB + BME denaturation and SDS-PAGE analysis can be performed as described earlier. After the SPOCQ reaction, the heavy chain band of Tras[HC]G4Y at 50 kDa shifts up slightly (lane II and III) and can be measured to have fluorescence (absorbance at λ= 565 nm and excitation at λ=
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