Anti-AGO1a | Argonaute 1a (Oryza sativa)

Samples:

1 – 2 µl of PageRuler™ Prestained Protein Ladder and 3ul Magic Mark XP, 10 to 220 kDa
2- 30 ug of Oryza sativa panicle, wild-type.
3- 30 ug of Oryza sativa panicle, ago1a mutant.

30 µg/well of total protein extracted 14 months ago and stored at -80C without freeze-thaw from rice young panicle tissues. Exact buffer components were: 100mM Phosphate pH8, 150mM NaCl, 5mM EDTA, 5mM EGTA, 0.1% Triton X-100, 1mM PMSF, cOmplete Protease Inhibitor tablet, Phosphatase Inhibitior 2, 3 & MG-132 and denatured with NuPage LDS Sample Buffer (Invitrogen) supplemented with 50mM DTT at 70°C/10 min. Samples were separated at RT on NuPAGE 3-8% Tris-Acetate gels and blotted for 16h on PVDF membrane (pore size of 0.45 um), using: wet transfer in the cold (30V). Blot was blocked with 5 % milk in TBS-T for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:2000 for 16h/4°C with agitation in 2% milk in TBS. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 in 2% milk in TBS-T for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: ThermoScientific SuperSignal West Femto Maximum Sensitivity Substrate. Exposure time was 15 seconds.