Cbb r 250

SDS-PAGE of crude cell extracts was carried out according to Laemmli (1970) (link). 7.5% SDS-PA gels were prepared according to a standard protocol. Proteins were visualized with colloidal Coomassie Brilliant Blue (CBB) R-250 (Serva, Heidelberg, Germany) or transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Vienna, Austria) for Western-blot analysis. Polyclonal rabbit antisera raised against the recombinant S-layer proteins TfsA (α-TfsA) and TfsB (α-TfsB) (Sekot et al., 2012 (link)) were used as primary antibodies in combination with a monoclonal goat α-rabbit secondary antibody labeled with IRDye 800CW (LI-COR Biosciences, Lincoln, NE, United States). S-Layer protein bands were visualized at 800 nm using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Polyacrylamide gel electrophoresis in the presence of SDS (SDS PAGE) was performed according to the method of Laemmli [27 (link)]. Aliquots of non-reduced or reduced extracts corresponding to 10 μg of protein content were loaded per lane. Proteins were separated in 12 % polyacrylamide gel and visualized by Coomassie Brilliant Blue (CBB-R250, Serva, Heidelberg, Germany) staining or electrophoretically transferred onto PVDF membrane (Immobilon-P Transfer Membrane, Millipore, Bedford, USA), essentially according to the method of Towbin et al. [28 (link)]. Molecular masses of the proteins were estimated by comparison with the Precision Plus Protein Standard Dual colour (BioRad, Prague, Czech Republic) running in parallel.