Page 129 - Tyrosine-Based Bioconjugations - Jorick Bruins

Inducible, Selective Labelling of Proteins via Enzymatic Oxidation of Tyrosine
Materials - SDS-PAGE
For SDS-PAGE (polyacrylamide gel electrophoresis) analysis a suitable system is required, e.g. Bio-Rad Mini-PROTEIN® Tetra Vertical Electrophoresis Cell. Either cast SDS-PAGE gels by hand using Bio-Rad protocol bulletin 6201 or purchase pre-cast gels from commercial vendors. Bio- Rad dual colour ladder standard is an advised reference protein ladder.
1. Sample buffer (2x) SB: Mix 4 mL glycerol (100%), 0.66 mL 1.0 M Tris(hydroxymethyl)aminomethane (Tris).HCl pH 6.8, 0.2 gram sodium dodecyl sulfate (SDS), 1 mg bromophenol blue and 5.44 mL MilliQ. Before use, add β-mercaptoethanol (BME) for the reduction of disulphide bonds (19:1 (v/v) SB:BME).
2. Staining solution: dissolve 1 g of Coomassie Brilliant Blue R-250 in 1 L solution of 5:4:1 (v/v/v) methanol (MeOH):water:acetic acid (AcOH).
3. Destaining solution: 5:4:1 (v/v/v) MeOH:Water:AcOH.
4. SDS-gel scanner, e.g. BioRad ChemiDocTM system.
Materials - HPLC/MS
High-performance liquid chromatography (HPLC) (and LC-MS) analysis can be used for the analysis of products obtained from the SPOCQ reaction, though protocols can vary for each different protein with regards to the used machine, eluents and columns. A full overview is beyond the scope of this protocol, only antibody analysis will be described in detail as an example.
1. HPLC system, e.g. Agilent 1220 HPLC Infinity system with in-line diode-array detector (DAD) (see Note 10).
2. RP-HPLC column for antibody analysis, e.g. ThermoScientificTM MAbPacTM reversed phase (RP) column (3.0 x 50 mm, 4 μm) column.
3. HPLC buffer A: 95% MiliQ, 5% MeCN with 0.1% trifluoroacetic acid (TFA).
4. HPLC buffer B: 95% MeCN, 5% MiliQ with 0.1% TFA.
5. Tris base for DTT denaturation: 0.1 M tris base buffered to pH 8.0 with 1.0 M HCl.
6. Freshly prepared 0.2 M dithiothreitol (DTT) in 0.1 M tris pH 8.0.
Methods - SPOCQ
SPOCQ should be performed at low temperature (4 °C), preferably in PBS buffer pH 5.5 and can be performed at any volume necessary. Consider the final volume to be 10 volume equivalents.
1. Add 8 volume equivalents of 10-200 μM of a C-terminal G4Y-expressed protein in PBS (pH 5.5) to a 1.5 mL Eppendorf tube and cool to 4 °C.
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