6 ml resource q column

Sca was purified from the supernatant of P. digitatum Sca^op strains (10⁶ conidia/mL) grown in P. digitatum minimal medium (PdMM) [50 (link)] after 11 days of growth at 25 °C with strong aeration. Cell-free supernatant was collected by centrifugation and dialyzed (2K MWCO, Sigma-Aldrich, St. Louis, MO, USA) against 20 mM phosphate buffer at pH 6.8. Given the predicted chemical properties of Sca (pI = 4.54), the dialyzed solution was applied to an AKTA Purifier system equipped with a 6 mL RESOURCE Q column (GE Healthcare, Chicago, IL, USA) equilibrated in the phosphate buffer. Elution was set with a linear NaCl gradient from 0 to 1 M in the same buffer. Surprisingly, Sca protein was not adsorbed in the resin and was present (as the major protein) in the flow-through after chromatography (Supplementary Figure S5). Thus, the Sca-containing flow-through was collected, dialyzed against Milli-Q water and lyophilized. Protein concentration was determined spectrophotometrically (A₂₈₀) considering the Sca molar extinction coefficient (Ɛ₂₈₀ = 2.43). The purity was monitored by SDS-PAGE using SDS-16% polyacrylamide gels calibrated with pre-stained protein size-standard SeeBlue^® and Coomassie blue staining. AfpB protein production, purification, and quantification were achieved as previously described [16 (link)].

Cell harvesting, lysis and NiNTA purification followed the same protocol as for Tcp12 and PCP₆. After NiNTA chromatography, the fractions containing protein were pooled and dialysed overnight into anion exchange buffer A (AEX) (20 mM Tris HCl pH 8.0, 50 mM NaCl). Subsequently, AEX chromatography was performed (Äkta, GE Healthcare, 6 mL ResourceQ column). Protein was loaded using AEX buffer A and eluted by applying a gradient from 0–50% AEX buffer B over 20 column volumes (20 mM Tris HCl pH 8.0, 1 M NaCl). As a final purification step SEC was performed using the same buffer as for Tcp12 and PCP₆. All proteins were flash frozen and stored at –80 °C.