Alexa fluor 647 azide

The reaction contained 100 mM potassium phosphate (pH 7.5), 121 μM Alexa Fluor® 647 azide (AF647, compound 3, Fig. 2, Click Chemistry Tools, 1299), 1.5 μM cetuximab either modified by mTGase or acylated as aforementioned, and incubated at 37°C for 3.5 hours. To remove excess unreacted reagents, each reaction mixture was desalted using 30 kDa MWCO centrifugal filters into 100 mM potassium phosphate (pH 7.6) prior to analysis.

All chemicals required for the synthesis of DA^yne, preparation of buffers, mushroom tyrosinase (Lot#: SLBZ0022), NBT, Amicon ultra 0.5 mL centrifugal filters (3k) and click reactions were purchased from Sigma-Aldrich unless otherwise stated. NMR solvents were obtained from Cambridge Isotope Laboratories Inc. SH-SY5Y neuroblastoma cells were acquired from ATCC. Cell culture media, additives, and consumables were purchased from Corning. High capacity streptavidin agarose resin, Zeba Spin Desalting Columns (7K MWCO, 0.5 mL), PDIA3 (ERp57) antibody (Cat. no. CL2444), and DAPI were obtained from Thermo Scientific. alamarBlue HS reagent and ProLong Glass were purchased from Invitrogen. BCA kits and C-18 spin columns were bought from Pierce. Mini-PROTEAN TGX Precast Gels, 0.2 μm nitrocellulose, and filter paper transfer stacks were obtained from BioRad. Biotin azide, THPTA, Alexa Fluor 647 azide, and azide agarose beads were purchased from Click Chemistry Tools. REVERT total protein stain kit, Odyssey TBS blocking buffer, and IRDye800CW streptavidin were purchased from LI-COR. Recombinant PDIA3 and PDI inhibitor screening kit (fluorometric) were obtained from BioVision (Cat. no. 7601–100 and K840–100, respectively).