Tris glycine precast sds page gels

Proteins were loaded onto 10% Tris–glycine precast SDS-PAGE gels (BioRad, Marnes-la Coquette, France) and stained with Coomassie Blue. The molecular mass under denaturing conditions was determined with PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, IL). The protein concentrations were determined by adsorption at 280 nm using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific) with theoretical molecular masses and molar extinction coefficient derived from the sequences (49,640 and 39,545 M⁻¹ cm⁻¹ for LPMO-FL and LPMO-CD, respectively, measured at 280 nm in water).

Filtered culture supernatant was loaded onto a 20-mL HiPrep DEAE FF 16/10 column (GE Healthcare), equilibrated with Tris–HCl 50 mM pH 7.8. Proteins were eluted using a linear gradient of 1 M NaCl (0 to 500 mM in 180 mL). Fractions containing recombinant proteins were pooled and concentrated. A fraction of eluate was loaded onto 10% Tris–glycine precast SDS-PAGE gels (BioRad) that were stained with Coomassie Blue. The protein concentrations were determined by absorption at 280 nm using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific) and theoretical molecular weights and molar extinction coefficient.

Filtered culture supernatant was loaded onto a 5-mL HisTrap HP column (GE Healthcare, Bus, France) equilibrated with buffer A (Tris–HCl 50 mM pH 7.8, NaCl 150 mM, imidazole 10 mM) that was connected to an Äkta purifier 100 (GE Healthcare). (His)₆-tagged recombinant proteins were eluted with buffer B (Tris–HCl 50 mM pH 7.8, NaCl 150 mM, imidazole 500 mM). Fractions containing recombinant enzymes were pooled, concentrated, and dialyzed against sodium acetate buffer 50 mM, pH 5.2. A fraction of eluate was loaded onto 10% Tris–glycine precast SDS-PAGE gels (BioRad) that were stained with Coomassie Blue. The protein concentrations were determined by absorption at 280 nm using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific) and theoretical molecular weights and molar extinction coefficient.