Strepmab classic hrp conjugate

Aβ oligomers were spotted onto nitrocellulose membranes and dried for 20 min at room temperature. The membrane was blocked with 4% (w/v) skimmed milk in TBST for 1 h at room temperature. After washing three times with TBST, the membrane was incubated with 200 nM or 10 nM of PrP–ALP in TBST containing 1 mM MgCl₂ and 100 μM ZnCl₂ for 1 h at room temperature. After washing, the membranes were rinsed with TBST containing 1 mM MgCl₂ and 100 μM ZnCl₂ for 5 min at room temperature. Chemiluminescent signals were detected using LAS 4000 mini with CDP-Star ready-to-use substrate (Roche Diagnostics, Basel, Switzerland). To evaluate the binding of PrP-strep tag II, StrepMAB-Classic HRP conjugate (IBA Lifesciences, Goettingen, Germany) was used for detection.

A plasmid expressing a 10-amino-acid fragment of the SitC Lpp with a C-terminal strep epitope under the control of the strong constitutive promoter P_tuf was constructed in the shuttle vector pLI50. The plasmid pLI50-sitC10AA was transformed into various RN4220 strains, and cultures were grown to early log phase (OD₆₀₀ = 0.5). Bacterial pellets were obtained by centrifugation, washed once with PBS, and resuspended in buffer (10 mM Tris-HCl [pH 8.0]) containing 50 μg/ml of lysostaphin. Samples were incubated for 15 min at 37°C before being quenched with 4× SDS-PAGE loading buffer. Samples were then heated at 70°C for 15 min before being clarified by centrifugation (18,000 × g, 5 min). Aliquots of supernatant were loaded onto an 18% Tris-tricine minigel and separated by electrophoresis using the Tris-tricine running buffer system (93 (link)). Protein was transferred to a nitrocellulose membrane (0.2 μM) and developed with an HRP-anti-strep tag conjugate as instructed by the manufacturer (StrepMAB-Classic HRP conjugate; IBA Life Sciences).