ACC synthase 7 | 1-aminocyclopropane-1-carboxylate synthase 7

10 ug of total protein extract from Nicotiana benthamiana leaves expressed ACS7­His­GST (1), 5 ug of purified ACS7­His­GST from N. benthamiana ACS7­His­GFP fusion protein transiently expressed in N. benthamiana leaves was purified on Ni ‐ NTA resin(2) and (3). Samples were separated on 10% SDS­PAGE and transfered to PVDP membrane by semi­dry blotting (1h). Blot was blocked with 3% skim milk in PBS­T (0,01% Tween 20) for 1h in RT with agitation. Then blot was incubated with primary antibody anti ACS7 at dilution of 1:500 in PBS­T for 1h in RT with agitation. After washes (3 times for 5 min. in PBS­T) blot was incubated with secondary antibody (anti­rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) at dilution 1:25000 for 1h in RT. The blot was washed 5 times for 5 min. with PBS­T and developed for 2 min with ECL­Plus according to the manufacturer's instructions. Exposure time was 3 min.

Courtesy of Dr. Agata Cieśla, Ludwików's lab, UAM, Poland