KrP | Kelch repeat-containing protein / galactose oxidase (At3g07720)

15 µg of total protein from Arabidopsis thaliana Col-0 root or shoot grown on plates with (+) or without iron (-) extracted and denatured with 2X Laemmli buffer (100 mM Tris pH 6.8; 12 % glycerol, 0.01 % bromophenolblue, 2 % β-mercaptoethanol, 4 % SDS) at 95°C for 5 min followed by 5 min on ice. Samples were separated on 4-20 % Mini-PROTEAN TGX Stain-Free gel and blotted 60 min to nitrocelulose membrane using wet transfer. Blots were blocked with 5% milk powder dissolved in TBST for 30 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2000 for 60 min at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed three times for 15 min in TBST buffer at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera) diluted to 1:4000 in for 60 min at RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent, using the FluorChem Q (protein simple) detection system. Exposure time was 3.2 seconds.