Anti ha hrp

Western blot analysis was used to confirm the presence of proteins in N. benthamiana during cell death assays. Three leaf discs were taken at 2 dpi, flash-frozen in liquid nitrogen, and ground to a fine powder using a micropestle. Leaf powder was mixed with 300 μl of plant protein extraction buffer (GTEN (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % v/v glycerol), 10 mM DTT, 2% (w/v) PVPP, 0.1% Tween–20, 1 x plant protease inhibitor cocktail (Sigma)). The sample was clarified by centrifugation (20,000 × g for 2 min at 4 °C, twice) and 40 μL of the resulting supernatant was added to 10 μL SDS-PAGE loading dye (RunBlue 4 x LDS sample buffer (Expedeon)), followed by incubation at 95 °C for 5 min.
Samples were subjected to SDS-PAGE/western blot analysis to detect epitope-tagged proteins. Pikp-1, Pikp-2, and AVR-Pik effectors were detected by probing membranes with anti-FLAG-HRP (Generon, 1:10,000 dilution), anti-HA-HRP (Thermo Fisher Scientific, 1:3000 dilution) and anti-Myc-HRP (Santa Cruz, 1:5000 dilution) antibodies, respectively, and LumiBlue ECL Extreme (Expedeon). Membranes were also stained with Ponceau S to observe protein loading.

A total of 4 96-well plates of clones were picked from two different nanobody libraries. All clones showing signal above background were sequenced. Six clones were both identified multiple times and found to be positive in multiple assays. These six clones were expressed and purified from 3 L bacterial cultures. A portion of each purified nanobody was biotinylated with 20-fold molar excess of NHS-biotin (Pierce) and 8 point, 5-fold, binding titrations were done for each clone using streptavidin-HRP or anti-HA-HRP (Thermo Fisher).