Horseradish peroxidase conjugated goat anti mouse secondary antibody

Protein was isolated from leaves of transgenic Arabidopsis in a protein extraction buffer (100 mM Tris-HCl, 20% glycerol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 200 mM DTT) and then boiled for 10 min and centrifuged at 12,000 g at 4 °C for 1 min. Total protein with the same volume was loaded onto SDS–PAGE gels, transferred onto PVDF blotting membranes, and then probed with the appropriate primary anti-GFP antibody (1:3000, Clontech) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:3000, Promega). The signal was detected using an imaging device (Tanon 5200).

Total proteins of roots were prepared by grinding on ice with an extraction buffer consisting of 50 mM Tris, 5% glycerol, 4% SDS, 1% polyvinylpolypyrrolidone, and 1 mM phenylmethylsulfonyl fluoride (pH 8.0), followed by 14,000 × g centrifugation at 4°C for 15 min. Fifteen milligrams of total protein were separated by electrophoresis on a 12% SDS–polyacrylamide gel and blotted onto polyvinylidene difluoride membranes, which were then probed with the appropriate primary anti-HA antibody (1:1000) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:3000, Promega). Protein levels were visualized using an ECL Kit (GE healthcare, USA).