SAM1-4 | S-adenosylmethionine synthase (affinity purified)

43.2 | 45 kDa (Arabidopsis thaliana)

Application example

70 μg of total protein from Arabidopsis thaliana wt Col-0, SAM3 knockdown, SAM1-4 knockdown, and one oeSAM3-YFP lines extracted with mortar and pestle using 2xSDS loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 0.02% bromophenol blue, 200 mM DTT), and denatured in the same buffer at 95°C for 10 min. Samples were separated on 11% SDS-PAGE  and blotted for 1h to PVDF using tank transfer. Blots were blocked with 5% milk powder in TBS-T overnight in a cold room with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed five times for 4 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG HRP-conjugated, from Agrisera) diluted to 1: 20 000 in  for 1h at RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent. The proteins were detected using CCD Image Fusion Fx7 after 30 seconds exposure time.

Courtesy of Louis-Valentin Meteignier, LGBP, Faculté des Sciences de Luminy, France