Figure 2.

[2+2+2] cycloaddition bioconjugation. A) Experimental proof-of-concept experiment linking the GFP-containing pDPrAF with a Fluor 488-Alkyne label. Successful cycloaddition reaction yields an isoindole ring linkage. B) SDS-PAGE analysis of the successful bioconjugation. The gels were analyzed first for fluorescence (top gel) and then stained with Coomassie blue to visualize protein (bottom gel). Lane L: Protein ladder. Lane 1: Wild-type GFP protein containing Tyr at residue 151 demonstrating no fluorescent signal after denaturation of the protein. Lane 2: GFP containing 1 at residue 151 that was not subjected to reaction conditions. Lane 3: GFP containing 1 at residue 151 was reacted in the presence of both the Fluor 488-Alkyne and the rhodium system, affording the cycloaddition product and fluorescence due to the covalent linkage of the fluorophore to the protein. Lane 4: GFP containing 1 at residue 151 reacted in the presence of the Fluor 488-Alkyne but no rhodium, demonstrating that non-covalent association of the dye does not occur.