Mini protean tgx stain free sds page gels

Fungal cells were grown on YMS plates for three to four days, harvested and washed in 1x PBS. Cells were resuspended in three volumes of high-salt extraction buffer (25 mM HEPES-NaOH [pH 7.9], 700 mM NaCl, 0.1 mM Na-EDTA [pH 8.0], 0.2% NP-40, 20% glycerol, 1.5 mM MgCl₂, 1 mM PMSF, 1 mM DTT, 1 μg/mL Leupeptin, 0.1 μg/mL Pepstatin) and incubated on ice for 10 min. Samples were sonicated (Branson Sonifier-450) for three sets of 10 pulses (output = 2, duty cycle = 80), keeping the sample on ice between sets. Insoluble material was removed by centrifugation at 14,000 rpm and 4°C for 10 min. Protein concentrations were quantified with a Qubit fluorimeter (Invitrogen). Total protein (75 μg per lane) was loaded onto pre-cast 4–20% gradient Mini-PROTEAN TGX Stain-Free SDS PAGE gels (BioRad). Proteins were transferred onto nitrocellulose membrane (0.2 μm pore size; BioTrace) by wet transfer (1 h, 110V). The following primary antibodies were used: ⍺H3 (Active motif 39763, mAb, 1:5000), ⍺H4K20me1 (abcam 9051, pAb, 1:1000), ⍺H4K20me3 (Active motif 39180, pAb, 1:2000). The following secondary antibodies were used: IRDye 680RD Goat anti-Mouse IgG (LI-COR 926–68070, 1:5000), IRDye 800CW Goat anti-Rabbit IgG (LI-COR 926–32211, 1:5000). Fluorescent signals were detected using a LI-COR Odyssey CLx imager.

To assess binding to chitin, 3.0 μM AfAA11A was incubated with 0.2% (w/v) α-chitin or β-chitin in 50 mM bis–Tris/HCl buffer, pH 6.5, at 30 °C and 1000 rpm. At different time points, the insoluble substrate was removed by filtration using a 96-well 0.2 μm PES filter plate (Millipore) installed in a vacuum manifold. The amount of LPMO remaining in solution was visualized on Mini PROTEAN TGX Stain-Free SDS-PAGE gels (Bio-Rad) with a Gel Doc EZ Imager (Bio-Rad) based on fluorescence signals and quantified with the Image Lab software, version 6.0.0. Solutions with known concentrations of AfAA11A were used as standard.