Mini protean tetra electrophoresis cell

The filter paper (FP), EG, CBH, and BGL activities of T. reesei were measured as described before (Ghose, 1987 (link)) by using Whatman No. 1 FP (Whatman, UK), CMC–Na (Sigma, USA), p-nitrophenyl-β-D-cellobioside (pNPC; Sigma, USA), and p-nitrophenyl-β-D-glucopyranoside (PNPG; Sigma, USA) as the substrates, respectively. One unit of enzyme activity was defined as the amount of enzymes releasing 1 mole of reducing sugar (or p-nitrophenol in the CBH and BGL assays) per minute under the assay conditions. Renaturing SDS–PAGE electrophoresis was performed in 12% polyacrylamide separating gel containing 0.1% SDS with 0.3% CMC–Na as the substrate; this procedure was performed in a Mini-PROTEAN tetra electrophoresis cell (Bio-Rad Laboratories, Milan, Italy).

Two different lots of the Calbiochem PSA (D10010682 (lot a) and D00116361 (lot b)) CS, as well as, the Scripps and Fitzgerald PSA CS were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R&D assay PSA calibrant (R&D, #1344-SE), prepared according to manufacturer recommendations, was also examined (Fig. 3). SDS-PAGE was performed according to Laemmli13 (link) with the below modifications. Samples (3 μg) were diluted in a 1 to 1 ratio with buffer (Bio-Rad Laboratories, #161–0737) under reducing conditions with 5% β-mercaptoethanol. PSA samples and 10 μL of protein molecular mass standards (Bio-Rad, #161–0137) were boiled for 5 minutes. After heating, samples were loaded on a 1.0 mm thick, 12.5%T/3.3%C polyacrylamide gel. Gels were run in a Mini Protean Tetra Electrophoresis Cell (Bio-Rad, #165–0827) and a voltage of 80 V for 15 min followed by 80 min at 120 V was applied. Proteins were silver stained and destained (Thermo, #24612) according to manufacturer recommendations.