Goat anti rabbit igg horseradish peroxidase hrp conjugate

Plants were grown along the surface of agar plates (pH 5.8) for 4 d and the plants were then transferred to agar plates at pH 4.3 or 5.8 and grown for an additional 3−5 d. Root tips (approximately 5 mm) were excised and homogenized in ice-cold homogenization buffer containing 50 mM MOPS-KOH (pH 7.5), 2.5 mM EDTA, 100 mM NaCl, 2 mM phenylmethylsulfonyl fluoride (PMSF), 20 μM leupeptin, 10 mM NaF and 1 mM dithiothreitol (DTT) using a mortar and pestle. The homogenate was centrifuged at 14,000 r.p.m. for 1 min and the supernatant (root extract) was solubilized by adding a half volume of SDS sample buffer containing 4.5% SDS, 30% sucrose, 22.5% β-mercaptoethanol, 0.018% Coomassie Brilliant Blue, 4.5 mM EDTA and 45 mM Tris−HCl (pH 8.0). Immunoblotting of the catalytic domain and the phosphorylated penultimate threonine residue of the PM H⁺-ATPase were performed as described previously (Inoue et al. 2011 (link)) with specific antibodies (Hayashi et al. 2010 (link)). The secondary antibody was goat anti-rabbit IgG−horseradish peroxidase (HRP) conjugate (Bio-Rad). The chemiluminescence signal was detected using a LightCapture system (ATTO). The signal intensities were analyzed using ImageJ software.