His tag 6xhis monoclonal antibody

Five hundred mL cultures of C. bescii strains (JWCB017, 049, and 054) were grown to mid-log phase at 75 °C, harvested by centrifugation at 6000×g at 4 °C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4× freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (75 µg) were electrophoresed in 4–15 % gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.

Five hundred mL cultures of C. bescii were grown to mid-log phase at 65, 70 or 75°C, harvested by centrifugation at 6,000×g at 4°C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4X freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (80 µg) were electrophoresed in 4–15% gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5,000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.

Clostridium thermocellum strains (JWCT02, JWCT06, JWCT07, and JWCT08) were grown to mid-log or stationary phase at 60 °C in 20 mL CTFUD-NY medium without uracil. Cells were harvested by centrifugation at 6000×g at 4 °C for 15 min, and cell pellets were washed using 50 mM Tris–Cl buffer (pH 8.0) and resuspended in Tris–Cl buffer to OD₆₀₀ 20. Cells were lysed by boiling in the presence of SDS [30 (link)]. Cell free extracts were electrophoresed in 4–15% gradient Mini-Protean TGX gels, that were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus and then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.