Extended Data Fig. 5: Development and optimization of a catalyst for P3PE crosslinking of amine-containing proteins.

a, Catalyst conception based on the use of a nucleophile (Nu) that can result in an intermediate with a good leaving group, and/or the use of Lewis acids (LeA) to facilitate the nucleophilic ring opening. b, Lewis acids compatible with our reaction condition are lithium and ammonium ions. c, Nucleophiles compatible with our reaction condition are those with high nucleophilicities and low basicity in a protic solvent environment, including pyridines, sterically hindered trisubstituted amines, and imidazoles. However, aliphatic amines and imidazoles can lead to failed crosslinking due to side reactions. d–f, Reducing SDS–PAGE for screening and confirming catalytic activities. The tested reaction conditions are listed on the right. d, Screened catalyst candidates chosen based on rationales described in a–c using reducing SDS–PAGE. e, Confirmation of catalytic and non-catalytic effect of pyridine and lithium on SPEARs formation, respectively. f, Exploration of pyridine’s catalytic effect under various conditions. All experiments were repeated three to six times with similar results. g–j, Optimization and characterization of pyridine-catalyzed formation of SPEARs. g–i, Reducing SDS–PAGE for optimization and characterization of pyridine-catalyzed P3PE-crosslinking reaction. The tested reaction conditions are listed on the right. g, Titration of pyridine concentration in the reaction mixture. h, Effect of antibody–Fab complex concentration and P3PE concentration on the overall yield of SPEAR formation. i, Time course of pyridine-catalyzed P3PE-crosslinking reaction. All experiments were repeated thrice with similar results. j, Comparison of ThICK-staining quality with ChAT SPEARs and calretinin (Calret) SPEARs with and without the use of pyridine. Color scale bar: pixel intensity. Experiments were repeated twice, representative image shown.