Anti-AGO5d | Argonaute 5d (Zea mays)

Samples from left to right:
Marker
Zea mays leaf (negative control), wild type (W23)
Zea mays anther, wild type (W23)
Zea mays ovary, unfertilized, wild type (W23)
Marker

Apparent MW of AGO5d: 130 kDa

Protein was extracted from >50mg of Zea mays anthers (0.2-0.4mm), ~200mg leaves (mature leaf), and ~300mg unfertilized ovaries with protein extraction buffer1. Proteins were separated on 4-12% NuPage LDS-PAGE (150V, ~120 min) and transferred overnight (30V, ~17 hr) to PVDF membrane (Invitrolon™ PVDF/Filter Paper Sandwiches, 0.45 µm, 8.3 x 7.3 cm), using wet transfer. Blot was blocked with 5% milk in TBS-T for 1 hour at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:250 with 2% milk in 5mL TBS-T for 1 hour 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 h at RT with agitation. The blot was washed 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 30 seconds.