SDS-PAGE and Immunoblotting of Whole-Cell Lysates

Whole-cell lysate analysis via SDS-PAGE and immunoblotting^{53 (link)} 1 ODU of cells was lysed using CelLytic B 2× Cell lysis reagent (Sigma-Aldrich, Saint Louis, MO, USA) and run on standard SDS-PAGE using 12% polyacrylamide gels with 5x reducing sample buffer (8 M Urea, 10% SDS). In total, 15 µL of each sample were loaded and run for 30 min at 200 V. SDS-PAGEs were blotted on nitrocellulose membranes using the iBlot^TM 1 gel transfer system (Invitrogen, Waltham, MA, USA), and His-tagged nanobody was detected using α-His mouse antibody (05-949, LOT: 3033896, 1:1000, Merck Millipore, Darmstadt, Germany) diluted in 2% skim milk, followed by incubation with a secondary HRP-conjugated rabbit α-mouse IgG (AP160P, 1:10,000, Merck Millipore, Darmstadt, Germany) diluted in TBS-T. Proteins were visualised using the Amersham ECL Prime Western Blotting System (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions and an Amersham gel imaging system.