Nupage 4 to 12 bis tris protein gel

Immediately before use, a 10 mM solution of DBCO-PEG₄-maleimide (Jena Bioscience) was prepared in anhydrous dimethyl sulfoxide and reacted, at a fourfold molar excess, with the protein of interest (i.e., eGFP, mCherry, or FGF-2) diluted to 0.1 mM in conjugation buffer [1× PBS with 1 mM EDTA (pH 7)]. The conjugation was reacted overnight at 4°C on a tube rotator. The next day, excess DBCO was removed, and the buffer was exchanged to 1× PBS (pH 7) using a 10-kDa Amicon Ultra-0.5 ml Centrifugal Filter (EMD Millipore). The DBCO-reacted protein-of-interest was then reacted, at a threefold molar excess, with an azide-terminated oligonucleotide label overnight at 4°C on a tube rotator. Reaction efficiency was assessed by running the product on a reducing SDS PA gel (NuPAGE 4 to 12% Bis-Tris Protein Gels, Thermo Fisher Scientific) and subsequently imaging the gel using a flat-bed fluorescent scanner (Typhoon 8600, Molecular Dynamics), probing for the fluorescent tag modifying the oligonucleotide label (fig. S11). In the case where overlabeling may be a concern, protein activity can be compared before and after conjugation. Protein-oligonucleotide conjugate was stored at −20°C until ready to use.