Immunoblotting of small and large proteins

For immune detection of small proteins after SDS-PAGE, samples were electroblotted onto PSQ 0.2 μm membranes (GE Healthcare, Munich, Germany) with 2 mA/cm² in a semi-dry system (transfer buffer 48 mM Tris, 39 mM Glycine, 20% methanol (v/v)). Larger proteins were electroblotted onto Nitrocellulose 0.45 μm membranes (GE Healthcare) with 750 mA for 2 h in a tank buffer system (transfer-buffer: 50 mM Tris, 384 mM Glycine, 20% Ethanol (v/v), 0.02% SDS (w/v)). Membranes were blocked with 5% milk powder in T-TBS buffer for at least 1 h. Polyclonal antibodies against the complete and SDS-denatured proteins Ffh, FtsY and YidC were raised in rabbits.⁸⁸ (link) Antibodies against the SecY peptide MAKQPGLDFQSAKGGLGELKRRC were raised in rabbits by GenScript Biotech (Leiden, Netherlands) and have been validated before.³⁰ (link)^,118 (link)^,121 (link)^,126 (link) Monoclonal peroxidase-conjugated antibodies against the His6-tag (HisProbe-HRP Conjugate) were purchased from Thermo Scientific (Langenselbold, Germany) and from Roche (Grenzach-Whylen, Germany). Peroxidase-coupled goat anti-rabbit antibodies (Caltag Laboratories, Burlingham, CA, USA) were used as secondary antibodies with ECL (GE Healthcare, Munich, Germany).