Quantifying FICD-Mediated BiP AMPylation

5 μM unmodified BiP substrate was incubated at 25°C with FICD proteins at the indicated concentration for the indicated time (Fig 3C and F) in a buffer consisting of HKM (25 mM HEPES‐KOH pH 7.4, 150 mM KCl and 10 mM MgCl₂) supplemented with 5 mM ATP. Reactions were quenched by the addition of LDS‐PAGE sample buffer and heating to 70°C. Samples containing 1 μg of BiP were loaded onto and resolved on a 4–12% SDS–PAGE gel and subsequently wet‐transferred onto a PVDF membrane. Total protein was imaged using Ponceau S stain (Fig 3C) or by use of parallel gels visualised with Coomassie protein stain (Fig 3F). The membrane was blocked with 1× ROTI Block (Roth) diluted in water and then probed for 1 h at 25°C with a mouse monoclonal IgG antibody reactive to AMPylated proteins (MoAb 17G6, Hopfner et al, 2020 (link)), diluted 1/1000 (v/v) in 1 × ROTI Block. The AMPylated BiP signal was imaged using an IRDye 800CW goat anti‐mouse IgG secondary antibody [Li‐Cor], diluted 1/2000 (v/v) in 1x ROTI‐Block.