Anti-AGO1b | Argonaute 1b (Oryza sativa)

Samples:

25 µg/well of Oryza sativa total protein from young panicle tissues. Exact buffer components were: 100 mM Phosphate pH8, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X-100, 1mM PMSF, cOmplete Protease Inhibitor tablet, Phosphatase Inhibitor 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 gel and blotted for 16h on PVDF membrane (pore size of 0.45 µm), 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 1:1000 for 1h/RT 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, AS09 602 Agrisera) 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 10-15 seconds.

Courtesy of M.Sc. Julie Pelletier, Meyers Lab, University of California, Davis, USA

Overview on antibodies with confirmed and predicted reactivity to rice proteins.