Irdye 800cw goat anti mouse igg h l antibody

To measure binding of prototoxin to MDCK.2 and ACHN cells the On-Cell Western assay was used as described previously [14] (link). Bound prototoxin was detected with mouse anti-Etx monoclonal Bio355 antibody (Bio-X Diagnostics S.P.R.L, Belgium) and IRDye 800CW goat anti-mouse IgG (H + L) antibody (LI-COR Biosciences, Lincoln, USA) at 1:500 dilution each. To quantify the amount of fluorescent signal, plates were imaged at 800 nm using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, USA). The binding activity of the mutant prototoxin was expressed as the percentage of fluorescence intensity relative to wild type prototoxin. To compare the means of the On-Cell Western assay data, Two-Way ANOVA analysis followed by Dunnett's multiple comparisons test was carried out using the GraphPad Prism 6 software (GraphPad Software, La Jolla).

Protein extraction buffer (10 mM Tris/Cl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche) was used for protein extraction from mycelia and plant materials. Protein samples of U. virens mycelia from the WT strain and the overexpression transformants were collected 7 days after culturing in potato sucrose (PS) liquid medium. Western blot analysis was performed with the eBlot™ L1 Protein Transfer System (GenScript) and Odyssey (Li-COR) detection system with IRDye 800CW Goat anti-Mouse IgG(H + L) antibody (926-32210, LI-COR,1:10000). Chemiluminescence detection in Fig. 4b was carried out with Horseradish Peroxidase (HRP)-conjugated Anti- HA antibodies (26183-HRP, Thermo scientific, 1:2000). For detection using this system, the membrane was first incubated with developing reagents (SuperSignal West Pico & West Femto), and then imaged using ImageQuant LAS 4000 (Life Sciences). Anti-HA-tag primary monoclonal antibody (M20003L, Abmart, 1:3000) was used to detect the expression of proteins in N. benthamiana leaves. Anti-GFP-tag primary monoclonal antibody (M20004L, Abmart, 1:3000) was used to detect the expression of SGP1 and its mutants in U. virens.

Protein and cell samples were boiled in 5 × SDS loading buffer (P10015L, Beyotime, Shanghai, China) at 100°C for 10 min, separated by 12.5% SDS-PAGE, and blotted onto nitrocellulose membranes (66485, PALL, Port Washington, NY, USA). The membrane was blocked for 1.5 h with 5% (w/v) skim milk (232100, BD Pharmingen San Diego, CA, USA) in PBS at room temperature. After washing three times with PBST (PBS containing 0.1% Tween 20), the membranes were exposed to primary antibodies in PBST for 1.5 h. The membranes were then washed three times using PBST. After 1 h of exposure to IRDye800CW goat anti-mouse IgG (H+L) antibody (926–32210, LI-COR, Lincoln, NE, USA), the membranes were washed and analyzed using the LI-COR Odyssey Imaging System (Li-Cor Biosciences). All experiments were performed at least in triplicate.