Anti-ACO1,2,3 | Aconitase

Brassica napus, Capsicum annuum, Cannabis sativa, Citrus sp., Cucumis sativus, Glycine max, Gossypium sp., Malus domestica, Manihot esculenta, Phaseolus vulgaris, Pisum sativum, Pinus pinaster, Populus sp., Solanum lycopersicum, Solanum tuberosum, Vitis vinifera

Samples:
1 - 50 ug of Arabidopsis thaliana whole leaf extract (vegetative leaves), 4 week-old plant
2 - 50 ug of Nicotiana benthamiana whole leaf extract (vegetative leaves), 4 week-old plant
3 - ~20 ug (due to low yields) of mitochondria isolated from 4 week-old Nicotiana benthamiana vegetative leaves *While samples in lanes 1, 2, 4, and 5 were freshly extracted, the sample in lane 3 was extracted and stored for 6 months at -20°C
4 - 50 ug of Vitis vinifera (grapevine) whole leaf extract (vegetative leaves)
5 - 50 ug of E. coli protein extract
Marker: MW (kDa) BLUelf prestained protein ladder

50 µg/well of total protein extracted freshly from A. thaliana leaves (1), N. benthamiana leaves (2), V.vinifera grapevine leaves (4) or E. coli (5). Exact extraction buffer components were: 2.5 mM Tris pH 8.2, 0.05 mM KCl, 20% glycerol, 400 mM sucrose, and 5 mM MgCl₂. Samples were denatured with SDS loading buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, 0.01% bromophenol blue) at 100°C for 10 min. Samples were separated at RT on 12% SDS-PAGE and blotted for 1h15 minutes to PVDF (pore size of 0.45 um) using wet transfer in the cold. Blot was blocked with 3% skim milk in PBS-T for 1h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 in PBS-T containing 1% skim milk overnight at 4°C with 60 RPM agitation. The blot was washed with 25 ml of 1X PBS-T five times at 5 minutes each with 60 RPM agitation. The blot was incubated with 1:8000 secondary anti-rabbit IgG, Horseradish peroxidase-linked antibody in PBS-T containing 1% skim milk for 2 hours with 60 RPM agitation. The blot was washed as described previously. The washed and drained blot was incubated with 2 ml of Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) for 5 minutes, and protein bands were exposed to X-ray films for 1 minute to capture signal.

Additional bands are a result of high protein load/well and concentrated primary antibody, which is suggested in our protocol, as it is easier to optimize it, once it is confirmed that the antibody is recognizing correct target protein.

Courtesy of Catherine Fust, PhD Candidate in the Department of Molecular and Cellular Biology, University of Guelph, Canada