Rabbit anti phospho p44 p42 mapk antibody

Leaf disks from 2- or 3-week-old plants were incubated in 5 mm MES buffer (pH 5.7) at room temperature overnight. After treatment with 200 μm ATP for 0, 5, 15, and 30 min, the samples were homogenized, and the total protein was extracted with protein extraction buffer (100 mm Tris-HCl [pH 7.5], 300 mm NaCl, 2 mm EDTA [pH 8.0], 1% Triton X-100, 10% glycerol, and protease inhibitor). The extracted proteins were mixed with 5× SDS loading buffer (10% SDS, 50% glycerol, 0.01% bromophenol blue, 10% β-mercaptoethanol, 0.3 m Tris-HCl [pH 6.0]) and boiled for 5–10 min. The total proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane. The membrane was then blocked in 5% skim milk in TBST buffer (50 mm Tris-HCl [pH 7.4], 150 mm NaCl, 0.1% Tween-20) and incubated with rabbit anti-phospho-p44/p42 MAPK antibody (Cell Signaling Technology, Danvers, MA, USA), followed by washing with TBST and incubation with HRP-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK).