Anti-AGO5a | Argonaute 5a (Z. mays)

Samples:

M– Marker
M – Marker
WT 1 – 72 µg of Zea mays seedling leaf extract
2 –72 µg of Zea mays premeiotic anthers extract from 12/16/24
3– 72 µg of  Zea mays unpollinated ovary extract from 12/16/24
M– Marker
ago5a mutant
4 – 72 µg of Zea mays ago5a adult leaf extract
5 – 72 µg of Zea mays ago5a premeiotic (0.2-0.5mm) anthers
M – Marker

Zea mays AGO5a molecular weight: 116 kDa

72 µg/well of total protein (extracted one day before running the gel and stored in -80C) from maize spikelet/ovary/leaf with SII buffer and denatured with exact buffer components at 70 °C/ 10 min. Samples were separated on 4-12% NuPage LDS-PAGE Tris-Acetate 3-8 % gel and blotted for 2 h at 30 V 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 overnight at 4C with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 with 5 % milk in 10 mL TBS-T for overnight/RT with agitation. The antibody solution was decanted and the blot was rinsed three times (5 min each) in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (Goat anti-rabbit IgG (H&L), HRP conjugated) diluted to 1:25000 in 2% milk in TBS-T for 1 h/RT with agitation. The blot was rinsed three times (5 min each) in TBS-T and developed with the Agrisera ECL SuperBright reagent. Exposure time was 5 seconds.