Anti-PIP2;7 | Plasma membrane aquaporin, N-terminal

Samples:
1 - Arabidopsis thaliana Col-0 of 11-day-old seedlings, phosphate (Pi) sufficiency
2 - Arabidopsis thaliana Col-0 of 11-day-old seedlings, with 5 days of Pi deficiency

5 µg/well of microsomal protein extracted from Arabidopsis thaliana roots. Microsomal protein was isolated using the Minute™ Plant Microsomal Membrane Extraction Kit (Invent Biotechnologies, MM-018) according to the manual instructions. The resulting pellet was solubilized with buffer containing 1% sodium 4-hexylphenylazosulfonate (Na-Azo, Excenen Pharmatech), 100 mM triethylammonium bicarbonate (TEABC, Sigma), pH 8.5, 2× Protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF), and denatured with buffer containing 1× NuPAGE™ LDS Sample Buffer (Invitrogen) and 100 mM dithiothreitol (DTT) at 70°C for 15 min. Samples were loaded into 4–12% Q-PAGE™ Bis-Tris Precast Gel (SMOBIO) SDS-PAGE and blotted to Immobilon®-P PVDF membrane (Millipore, pore size of 0.45 µm) for 1 h using wet transfer. Blot was blocked with 1% BSA in 1× PBS solution with 0.2% Tween 20 (PBST, pH 7.2) for: 1 h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5,000 in PBST containing 1% BSA for 1 h/RT with agitation. The antibody solution was decanted, and the blot was washed 4 times for 5 min in PBST at RT with agitation. Blot was incubated in matching secondary antibody (Goat anti-Rabbit IgG (H&L), HRP conjugated, AS0 602, Agrisera) diluted to 1: 25 000 in PBST containing 1% BSA for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 30 seconds.

Courtesy of Dr. Tzu-Yin Liu, National Tsing Hua University, Taiwan