Guanidine hcl

The mature form of mouse, human or bank vole (aa23-231; mouse numbering) PrP was cloned into pET41a expression vectors (EMD Biosciences) and expressed in E. coli Rosetta using the Express Autoinduction System (Novagen). Inclusion bodies were prepared using the Bug Buster reagent (Novagen) and solubilized in lysis buffer [guanidine-HCl 8 M (Sigma-Aldrich)], sodium phosphate 100 mM, Tris–HCl 10 mM, pH 8.0 (Sigma-Aldrich)) for 50 min at 23 °C and then centrifuged at 16,000×g for 5 min at 23 °C. Binding, refolding, and elution using an AKTA Explorer system has been described previously [30 (link)].

Heads or thoraces were sectioned from five 35-day-old flies and homogenized in 6 M guanidine HCl (Sigma); 10 mM TRIS (Sigma), pH 7.3, as previously described (Xu et al., 2008 (link)). ATP content was measured using an ATP determination kit (Molecular Probes, Eugene, OR). Protein abundance was measured using a Bradford protein assay, and ATP abundance was normalized to total protein abundance as previously described (Xu et al., 2008 (link)).

To generate biotinylated hyaluronan, 35 mg of high MW hyaluronan (R&D Systems, UK) was reconstituted at 1 mg/ml in MES buffer (0.1 M, pH 5; Sigma, UK) for 24 h at 4 °C with agitation. Sulpho-NHS (Sigma, UK), biotin hydrazide (Sigma, UK) and EDAC (Sigma, UK) were added to a final concentration of 0.184 mg/ml, 1 mM and 30 μM, respectively, and the reaction incubated at 4 °C overnight with agitation. The reaction was stopped by the addition of 1 ml of 4 M guanidine-HCl (Sigma, UK), and the resulting biotinylated hyaluronan (bHA) dialysed against 4 × 400 ml volumes of deionised H₂O using 10 kDa molecular weight cut-off Slide-a-Lyser dialysis cassettes (Pierce). The recovered final volume was ~40 ml, indicating a final concentration of ~0.83 mg/ml, which was confirmed by hyaluronan Quantikine ELISA (R&D Systems) carried out following manufacturer's instructions. Single-use aliquots were stored at −20 °C. This protocol has been described to yield approximately 1 biotin molecules per 93 disaccharides of hyaluronan (Frost and Stern, 1997). For in vivo use, mice were treated with 20 μg bHA diluted in sterile PBS by intranasal inhalation under isoflurane anaesthesia.

Human immunoglobulin (Ig) and anti-alpha3(IV)NC1 collagen Ab in serum and fusion cell supernatants were detected by ELISA, as described [20 (link)], using the following reagents: Human IgG and IgM standards (Thermo Scientific, Waltham, MA, USA), goat-anti-human Ig or goat-anti-human-kappa capture Ig, and alkaline-phosphatase conjugated goat anti-human Ig, anti-human IgG, and anti-human IgM (Southern Biotech, Birmingham, AL, USA). Results for binding to antigen [bovine alpha3(IV)NC1 collagen (Eurodiagnostica)] were recorded as OD on antigen minus OD on wells coated with diluent only (6 M guanidine-HCl, Sigma-Aldrich), using as controls mouse anti-alpha3(IV)NC1 collagen IgG (Mab3) and Goodpasture patient serum (Eurodiagnostica).