Slide a lyzer cassette

Silk fibroin aqueous solution was obtained from B. mori silkworm cocoons according to previously published literature (Di Buduo et al., 2018 (link)). Briefly, dewormed cocoons were boiled for 30 min in 0.02 M Na₂CO₃ solution at a weight to volume ratio of 10 g to 4 L. The fibers were rinsed for 20 min three times in ultrapure water and dried overnight. The dried fibers were solubilized for 4 hr at 60°C in 9.3 M LiBr at a weight to volume ratio of 3 g/12 mL. The solubilized silk solution was dialyzed against distilled water using a Slide-A-Lyzer cassette (Thermo Scientific, Waltham, MA, USA) with a 3500 MW cutoff for three days and changing the water a total of eight times. The silk solution was centrifuged at maximum speed for 15 min to remove large particulates and stored at 4°C. The concentration of the silk solution was determined by drying a known volume of the solution overnight at 60°C and massing the remaining solids.

Silk fibroin aqueous solution was obtained from Bombyx mori (CREA – AA), as previously described.^[35 (link)
^] Briefly, dewormed cocoons were boiled in Na₂CO₃. The fibers were rinsed in ultrapure water and dried overnight. The dried fibers were solubilized in LiBr. The solubilized silk solution was dialyzed against distilled water using a Slide‐A‐Lyzer cassette (Thermo Scientific) with a 3500 MW cutoff for three days. Before usage, the silk solution was centrifuged at maximum speed to remove large particulates.

The cleared lysate was loaded onto Ni-NTA resin (Superflow, IBA) which was preconditioned with water and 1x native lysisbuffer. Binding was performed overnight at +4 °C under continuous agitation. Next day, the resin was settled vertically and washed with 6 bed volumes of lysisbuffer containing 20 mM of Imidazole. Thereafter, the bound recombinant protein was eluted with 4 bed volumes of elution buffer (500 mM of NaCl, 50 mM phosphate buffer (pH 8.0), 5 % v/v glycerol and 300 mM of Imidazole). Subsequently, salts were removed by dialysis in a Slide-a-Lyzer cassette (3,500 MWCO, 3–12 ml capacity, ThermoScientific) for 48 h at +4 °C. The dialysis buffer (PBS complemented with 0.1 M NaCl and 5 % v/v glycerol) was refreshed three times. After dialysis the recombinant protein content was analyzed for protein concentration (BCA assay, ThermoScientific) and Western Blot. A second round of purification was performed by fast protein liquid chromatography (FPLC) with a 1 ml volume HisTrap-HP^TM column (GE Health Care life sciences) with a pressure flow of 0.15 ml/min. The his-tagged proteins were eluted from the column with 10 column volumes (CV) of a linear gradient ranging from 50 mM Imidazole up to 500 mM Imidazole in 0.5 ml aliquots. These were analyzed by Western blot and the eluates containing the his-tagged proteins were subsequently pooled and dialyzed as described above.