Guanidine hydrochloride gdnhcl

The ECOS™ BL21 (DE3) competent Cells were purchased from Yeasterm Biotech Co., Ltd. (Taipei, Taiwan). Lysogeny broth (LB) was purchased from Cyrusbioscience (New Taipei City, Taiwan). Isopropyl β-D-1-thiogalactopyranoside (IPTG), Triton X-100, guanidine hydrochloride (GdnHCl), trifluoroacetic acid, D₂O, 2,2-dimethyl-2-siapentane-5-sulfonate (DSS), and ampicillin were purchased from Sigma-Aldrich (St. Louis, MO, USA). ¹⁵N-labeled ammonium chloride was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Coomassie Brilliant Blue G-250 was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). Sodium azide was purchased from Merck (Millipore, Burlington, MA, USA).

The bacterial strains and vectors used in this work are listed in Table 1. L. lactis was used for expression of the modified peptides and cultured in M17 broth supplemented with 0.5% (w/v) glucose (GM17) or GM17 agar for genetic manipulation or in minimal expression medium (MEM) (Rink et al., 2005 (link)) for protein expression at 30°C.
Chloramphenicol and/or erythromycin were used at 5 μg/mL when appropriate.
The plasmids pNZnisA-E3 (Kuipers et al., 2004 (link)) and pNZnisA leader6H were used for the expression of nisin and as a template for the construction of the designed hybrid peptides. Plasmids pIL3BTC (Rink et al., 2005 (link)) and pIL3EryBTC (van Heel et al., 2013 (link)) encoding the nisin modification machinery were used to produce and modify the fusion peptides.
Commercial vasopressin [(Arg⁸)-Vasopressin acetate salt] was ordered from Sigma-Aldrich as a control. Endoproteinase Glu-C from Staphylococcus aureus V8 (V8), cyanogen bromide (CNBr), hydroxylamine (NH₂OH), 3-Bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BNPS-Skatole), and trypsin were ordered from Sigma-Aldrich as cleavage reagents. Ammonium acetate (CH₃COONH₄), formic acid, guanidine hydrochloride (Gdn-HCl), and lactic acid were ordered from Sigma-Aldrich. All the chemical reagents are of analytic purity.

Proteinase K (PK) and guanidine hydrochloride (GdnHCl) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reagents for enhanced chemiluminescence (ECL Plus) were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). Anti-tau mouse monoclonal antibodies RD3 and RD4 (Sigma-Aldrich) against human tau repeating region and sheep anti-mouse (SVM) IgG conjugated with horseradish peroxidase as a secondary antibody (AC111P, CHEMICON International, Inc, Burlington, MD) were used. Antibodies against Phospho-Tau (Thr231) and phospho-Tau (Ser396) were purchased from Cell Signaling Technology (Danvers, MA).