Anti-P2K1| L-type lectin-domain containing receptor kinase I.9

Samples:
Col-0 - Arabidopsis thaliana WT
dorn1-3 - Arabidopsis thaliana mutant
P2k1ox - Arabidopsis thaliana overexpressing line

15-20 µg/well of total protein extracted freshly from Arabidopsis thaliana microsomes. The exact buffer components were: 50 mM Hepes, pH 7.5, 250 mM sucrose, 15 mM EDTA, 5% glycerol, 1 mM DTT, and 1X HALT protease/phosphatase inhibitors. Protein concentration was measured using Bio-Rad Protein Assay Dye (cat # 5000006). SDS-PAGE samples were prepared by mixing 30 µL of protein extract with 10 µL 4x Laemmli buffer (0.25 M Tris HCl, pH 6.8, 8% SDS, 40% glycerol, 20% beta-mercaptoethanol, bromphenol blue) and heating at 95ºC for 5 min. SDS-PAGE and WB were performed and only low-molecular weight bands were visualized. Therefore, 20 µl of the prepared earlier SDS-PAGE samples were mixed with 30 µl of 2x Laemmli buffer with 8 M urea (8 M urea, 0.125 M Tris HCl, pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, bromphenol blue), so, the final urea concentration in the samples was 5.3 M. The samples were heated at 95ºC for 5 min, and SDS-PAGE was performed using 10 % acrylamide gel containing 8 M urea following blotting for 1.5 hrs to PVDF membrane (Immune-Blot PVDF Membranes for Protein Blotting, Bio-Rad, cat # 1620177, pore size of 0.2 µL), using wet transfer in the cold transfer buffer. The blot was blocked with 5 % dry non-fat milk for 1h/RT with agitation. The blot was incubated in the primary antibody at a 1: 1000 dilution ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. The blot was incubated in a matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 in TBS-T for 1 h/RT with agitation. The blot was washed as above and developed with the extremely low femtogram detection range chemiluminescent reagent. Exposure time was from 10 seconds to 1 minute.