LCY | Lycopene beta-cyclase (chloroplastic)

application example

Total protein from Arabidopsis thaliana leaves, wilde type and Lut2 (Arabidopsis thaliana mutant devoid of lycopene-epsilon-cyclase, an enzyme at the same branching point catalyzed by lycopene-beta-cyclase (LCY)) corresponding to 0.5 µg of chlorophylls, were extracted with loading buffer (10% glycerol, 62.5 mM Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol) and denatured at 100°C (boiling water) for 1 min.

Proteins were separated on 12% SDS-PAGE (Laemly) and blotted 1h to PVDF using tank transfer. Blots were blocked with blocking solution (PBS 1X, 0.2% w/v Tween, 5% powder milk) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody diluted in blocking solution, at a dilution of from 1:1000 to 1:128000) for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in blocking solution at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG alkaline phosphatase conjugated, diluted to 1:30 000 in blocking buffer for 1h at RT with agitation. The blot was washed 2 times for 10 min in blocking solution and once with PBS 1X solution for 10 min, then developed in developing buffer NBT/BCIP by manual agitation. 

Courtesy of Stefano Cazzaniga, University of Verona, Italy

Mutant devoid of LCY is not available (not viable) and using lut2 mutant allowed to test if the antibody discriminates the two different cyclases.