Anti-PI3K | Phosphatidylinositol 3-kinase VPS34

Samples:
1- MW Marker
2- Arabidopsis thaliana Wild type (Col-0) – Total Protein
3- Arabidopsis thaliana Wild type (Col-0) – Plasma Membrane
4- Arabidopsis thaliana Wild type (Col-0) – Inner Membrane
5- Arabidopsis thaliana Wild type (Col-0) – cytoplasm

Protein extracted freshly, from Arabidopsis thalianaWild Type (Col-0) plants grown on solid MS media for 2 weeks under long-day conditions. Extracted samples were mixed with 2x Laemmli sample buffer and bet-mercaptoethanol and denatured at 95°C for 5 min prior to SDS-PAGE analysis. Samples were separated on 4-20% SDS-PAGE at room temperature and blotted for 7 min to nitrocellulose Membrane (pore size of 0.2 µm), using: semi-wet transfer system. Blot was blocked with 1x TBS 1% Casein Blocker for 1h/RT. Blot was incubated in the primary antibody at a dilution of 1: 500 for ON/4°C with agitation. The antibody solution was decanted, then washed 3 times for 5 min in TBS-T at RT. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25k for 3h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: SuperSignal West Pico Plus chemiluminescent (ThermoScientific). Exposure time was 1 minute.