Vivaspin 500 centrifugal filter units

For antigen-specific immunoaffinity purification of anti-Dsg3 and anti-Dsg1 IgG, the entire ectodomains of Dsg3 and Dsg1, respectively, were immobilized on N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (GE Healthcare, Buckinghamshire, UK) as previously described (26 (link)). Immunoaffinity purifications were performed as follows. The immobilized protein matrix was transferred into microcentrifuge spin columns (Thermo Fisher Scientific, Darmstadt, Germany) and washed three times with tris-buffered saline supplemented with 5 mM CaCl₂ (Ca²⁺-TBS). The concentrated IgG of the PV patients was diluted 1:1 with Ca²⁺-TBS and incubated with the immobilized protein for 30 min at room temperature. The flow-through fraction was collected by centrifugation at 500x g for 30 s. After several washing steps with Ca²⁺-TBS (until OD₂₈₀ < 0.05) the anti-Dsg3 and anti-Dsg1 IgG fractions were eluted from the matrix with IgG elution buffer (Thermo Fisher Scientific) until the OD₂₈₀ was below 0.05, and immediately neutralized with 1M Tris pH 9.0. All eluted fractions were pooled and buffer was exchanged to PBS using Vivaspin 500 centrifugal filter units (Sartorius AG, Göttingen, Germany). Finally, Dsg3/1-depleted PV IgG (flow-through fractions) and anti-Dsg3 PV IgG (eluted fractions) were analyzed for anti-Dsg3 and anti-Dsg1 autoantibody reactivity by ELISA (Euroimmun).

Eluates from biotin pull-down assays were concentrated in Vivaspin 500 centrifugal filter units (Sartorius) and resolved by SDS-PAGE in 4–12% Bolt Bis-Tris precast gels (Invitrogen). Proteins were visualized with GelCode blue stain reagent (Invitrogen) and avidin monomers (∼15 kDa) and dimers (∼30 kDa), still present in the samples, as well as an endogenously biotinylated protein found at ∼130 kDa were excluded. For that, the gels were cut into the following slices: slice A1 from ∼15 kDa to ∼25 kDa; slice A2 from ∼30 kDa to 50 kDa; slice B from 50 kDa to ∼130 kDa, and slice C from ∼140 kDa to up to the gel. These four slices were then subjected to in-gel trypsin digestion as described previously (69 (link)). In brief, proteins were first reduced with DTT and then alkylated with chloroacetamide. Afterward, gel pieces were saturated with trypsin and incubated overnight at 37 °C. The resulting peptides were extracted from the gel, dried down in a vacuum centrifuge, and stored at –20 °C. Peptide mixtures were resuspended in 0.1% formic acid for LC-MS/MS analysis.