Extracellular Protein Precipitation Technique

Extracellular proteins (ECPs) were precipitated from cell-free supernatants by ammonium sulfate precipitation, as previously described [30 (link), 64 (link)]. Briefly, ammonium sulfate crystals (Fisher Scientific) were added to cell-free supernatants to achieve 65% saturation and incubated at 4 °C with gentle mixing for 24 h. Precipitated proteins were collected by centrifugation, resuspended in Tris buffer, and dialyzed twice against the same buffer in 10 Kda dialysis cassettes (Slide-A-Lyzer (Thermo Fisher)). Following dialysis, the volume of all protein samples were adjusted to 20 ml by the addition of cold Tris buffer. Protein concentration of each sample was determined by the Bradford assay (Pierce Coomassie Plus Protein Assay, Thermo Fisher). These concentrated proteins were used for all enzymatic assays.