Anti-AGO5c | Argonaute 5c (Zea mays)

Samples:
M – Marker
M – Marker
1 – 68 µg Zea mays WT premeiotic (0.5-1mm) anthers extract (TCA/acetone precipitation)
2 – 30 µg Zea mays ms28 premeiotic anthers (TCA/acetone precipitation)
3– 6 8 µg Zea mays ms28 premeiotic anthers (TCA/acetone precipitation)
M – Marker

Extracted amples were separated at RT using Tris-Acetate 3-8% NuPage LDS-PAGE gel, then blotted to PVDF membrane (0.45 µm pore size) using wet transfer O/N in the cold. The day after, blot was stained with Ponceau and dried down. Blot was blocked with 5% milk in TBS-T O/N at 4ºC with agitation followed by the incubation with the primary antibody at a dilution of 1:500 with 2% milk in 5mL TBS-T for 1 hr at RT with agitation. Blot was washed with TBS-T 3x at RT with agitation (5 min per wash). Blot was incubated in matching secondary antibody (Goat anti-rabbit IgG (H&L), HRP conjugated, AS09 602, Agrisera) diluted to 1:25 000 in 2% milk in TBS-T for 1 hr at RT with agitation and followed with washes with TBS-T 3x at RT with agitation (5 min per wash), then developed with the Agrisera ECL Bright and SuperBright reagents. Exposure time was 3 minutes.

Target protein band identity was confirmed by IP-MS analysis.