Anti-ABI5 | Abscisic acid insensitive 5 (anti-protein antibody)

30 µg (per lane) of total proteins extracted freshly from 3-day-old germinating seeds with the exaction buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM DTT, 1% (v/v) Triton X-100, Sigma protease inhibitor cocktail) and denatured with 4X SDS sample buffer at 95°C for 5 min. Samples were separated using 10% SDS-PAGE and transferred onto PVDF membrane (0.2 m pore size), using wet transfer. After blocking with 5% milk in PBS-T for 0.5h at RT (room temperature) with agitation, the blot was incubated in the primary antibody at a dilution of 1: 10, 000 (from the initial antibody solution at 1 µg IgG/µl) in PBS-T at 4°C with agitation. The antibody solution was decanted, and the blot was washed 4 times, each for 10 min, in PBS-T at RT with agitation. The blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, 1:20, 000) for 1h at RT with agitation. It was washed and developed with Agrisera ECL SuperBright.

Courtesy of Dr. Hong Wang, University of Saskatchewan, Canada