Samples: 1 and 2. pBA002 vector as negative control (technical replicates).
3 and 4. pBA002:AtACO4 (technical replicates).
Total protein extracts were prepared from Nicotiana benthamiana whole leaves agroinfiltrated with the indicated vectors.
18 µg/well of total protein extracted freshly from Nicotiana benthamiana with following buffer components: Tris-HCl pH 7.5 100 mM, Urea 8 M, Triton x-100 (0.2%), Sarkosyl (0.2%), PMSF 1mM, Pepstatin 1 µg/mL, Leupeptin (1 µg/mL), NEM 2 mM and Iodoacetamida 10 mM and denatured with 4x Laemmli Sample Buffer at 95 °C for 5 min. Protein samples were separated on 12% SDS-PAGE and blotted for 1h to PVDF/nitrocellulose (pore size of 0.2 µm), using semi-dry transfer. Blot was blocked with 3% milk in TBS-T for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 (anti-ACO4) or 1:2000 (anti-flag, Agrisera, No. AS15 3036, Lot. 1902) in 3% milk in TBS-T ON/4°C with agitation (anti-ACO4 and anti-FLAG in corresponding membrane). The antibody solution was removed and the blot was washed three times for 10 min in TBS-T at RT with agitation. Blot was incubated with Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) or ECLTM Peroxidase labelled anti-mouse antibody) diluted to 1:25 000 for 1h/RT with agitation. The blot was washed as above and developed for 30 min with AgriseraECLSuperBright. Exposure time was 60 seconds.
Asterisk denotes a band recognized in all samples: both in negative control as in ACO4 samples, whereas the second band is exclusive of the ACO4 samples with the same MW as the ACO4 samples with FLAG-tag. The second ban may be an orthologus protein in N. benthamiana.
Courtesy of Diana Fuertes Bailón (L.Maria Lois Lab), CRAG, Barcelona, Spain
