Anti-AGO1c | Argonaute 1c (Oryza sativa)

Samples:
1 – 2 µl of PageRuler™ Prestained Protein Ladder and 3ul Magic Mark XP, 10 to 220 kDa
2- 25 ug of Oryza sativa panicle, wild-type.
3- 25 ug of Oryza sativa panicle, ago1c mutant.

25 µg/well of total protein from Oryza sativa 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 1:2500 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 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.