Anti mouse igg conjugated to alkaline phosphatase

Proteins were extracted with buffer E (1:2 tissue to buffer ratio) and 15 μL of supernatants were separated by electrophoresis on a 12% SDS-gel and transferred by semi-dry blotting to a nitrocellulose membrane (0.45 μm pore size, Thermo Fisher Scientific). The membrane was blocked with 5% milk powder in TBS-T (20 mM TRIS-HCl, pH 7.6; 150 mM NaCl; 0.1% (v/v) Tween 20) and was incubated with custom-made rabbit polyclonal anti-XMPP antibody (1 μg mL⁻¹) in TBS-T with 0.5% (w/v) milk powder over night at 4 °C. The membrane was washed three times for 10 min with TBS-T and incubated with secondary goat anti-rabbit IgG horseradish peroxidase conjugated antibody (RABHRP1, 1:5000, Sigma-Aldrich) in TBS-T for 1 h at room temperature and washed as before. For detection, SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used. The chemiluminescence was detected by a Lumi-Imager F1 (Hoffmann-La Roche). For detection of YFP, membranes were incubated with monoclonal anti-GFP antibody from mouse (Roche 11814460001; clones 7.1 and 13.1; 1:15000 diluted). This antibody does not bind to mTFP1. Anti-mouse IgG conjugated to alkaline phosphatase (Sigma-Aldrich A3562; 1:10,000 diluted) was used as a secondary antibody.

The full-length VP3 and its fragments were expressed in E. coli BL21 (DE3) Star (Novagen) with a 6×His tag at the N-terminus after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Amresco). Recombinant VP3 (rVP3, 37 kDa) was expressed at 37 °C for 4 h and solubilized from inclusion bodies with 2 M urea. Larger and shorter VP3 fragments composed of 130 and 50 amino acids, respectively, were expressed at 20 °C for 18 h. The shorter VP3 fragments were tagged to the C-terminus of GFP generating approximately 37 kDa proteins. All fragments could be recovered from the soluble fraction.
The rVP3 and its fragments were purified by affinity chromatography in a Ni-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) and were eluted with 500 mM imidazole. SDS-PAGE and Western blot were performed to evaluate the steps of the expression and purification procedures using a monoclonal antibody anti-polyhistidine as the primary antibody (Sigma) and anti-mouse IgG conjugated to alkaline phosphatase as the secondary antibody (Sigma). The Western blot detection was performed with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Promega).