Pre cast 4 12 sds page gels

Pre-cast 4–12 % SDS-PAGE gels (Life Technologies, Carlsbad, CA) were used to visualize proteins bound and unbound to lignin extracted from corn stover. All gels were run at 200 V constant for 50 min in 3-(N-morpholino)propanesulfonic acid (MOPS)-SDS buffer. The desalted starting enzyme, supernatant containing unbound proteins, and the lignin pellet containing protein bound to insoluble lignin were diluted with 4×LDS sample buffer (3:1 sample:buffer) and held at 70 °C for 10 min in preparation for SDS-PAGE.

Pre-cast 4–12% SDS-PAGE gels (Life Technologies, Carlsbad, CA) for Cel7A and for CelA were used to visualize proteins bound and unbound to lignin extracted from corn stover. All gels were run at 200 V constant for 50 min in MOPS-SDS buffer. For binding studies, 300 μg of desalted protein was incubated with 6 mg of lignin, corresponding to a process loading of ~30 mg protein/g cellulose for a theoretical biomass containing 30% lignin and 50% cellulose. The protein/lignin combination was incubated at room temperature for 60 min in 25 mM sodium citrate buffer at pH 4.8 unless otherwise stated. After the incubation, the lignin was centrifuged at 20,000 × g for five min and the supernatant containing the unbound protein was collected while the lignin pellet was washed an addition four times with citrate buffer. The desalted starting enzyme, supernatant containing unbound proteins, and the lignin pellet containing protein bound to insoluble lignin were diluted with 4X LDS sample buffer (3:1 sample:buffer) and held at 80 °C for 10 min in preparation for SDS-PAGE.