Amersham ecl prime western blotting system

To determine whether the immunizations were stimulating an immune response, Western blots were conducted, which were timed after the third immunization (for the HVR group) and prior to the challenge with A. marginale St. Maries (StM). Proteins were separated by SDS polyacrylamide gel electrophoresis using 4–20% precast gels (BioRad, Hercules, CA, USA) and were transferred to polyvinylidene fluoride (PVDF) membranes, which were placed in blocking buffer and shaken for 1 h. Bovine serum was collected from each animal and then diluted to 1:200. A total of 100 µL of antibody was added, and the membrane was shaken for an additional 1 h. The membranes were washed according to the established protocol and incubated with a 1:20,000 dilution of anti-bovine Ig2:HRP for 1 h with shaking, and the membranes were detected using the Amersham ECL Prime Western Blotting System (GE Healthcare, Chicago, IL, USA).

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.