LUT1 | Beta-carotene hydroxylase
AS15 3084 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Total proteins from Arabidopsis thaliana leaves wilde type (left panel) and lut 1 mutant (right panel), corresponding to 1 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 15% 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 1: 1,500, 1:3,000, 1:6,000, 1:12,000 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
Alternative names: Cytochrome P450 97C1, Carotene epsilon-monooxygenase, chloroplastic.
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