Hrp conjugated anti gfp

Plant tissues were harvested at 2–3 dpi and immediately snap‐frozen in liquid nitrogen. The harvested samples were ground and mixed with 250 µl of 5 × SDS sample buffer (250 mM Tris‐HCl adjusted to pH 6.8, 10% sodium dodecyl sulphate [SDS], 0.1% [wt/vol] bromophenol blue, and 40% glycerol, supplemented with 50 mM dithiothreitol before use). The mixture was boiled at 96 °C for 10 min, vortexed vigorously, and centrifuged at 15,000 × g for 1 min. Fifteen microlitres of the supernatant was used for SDS‐polyacrylamide gel electrophoresis (PAGE) to separate proteins. Samples were run on an SDS‐polyacrylamide gel with 120 V for 80 min, electroblotted onto a PVDF membrane with 300 mA for 120 min and probed with anti‐FLAG (Sigma‐Aldrich) and anti‐mouse antibody conjugated to horseradish peroxidase (HRP) (Sigma‐Aldrich) or with HRP‐conjugated anti‐GFP (Santa Cruz). Visualization was achieved using SuperSignal West Pico and Femto substrate (Thermo Scientific) in ImageQuant LAS 500 chemiluminescence CCD camera (GE Healthcare). Lastly, the membrane was stained with Ponceau S (Sigma‐Aldrich) for 10 min to visualize total protein loading.