FER | Ferritin (plant)

10 µg of total protein from Arabidopsis thaliala whole seedlings, various cellular fractions, extracted with (see below*) were separated on 15 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 5% Non fat dairy milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3000 overnight at 4°C with agitation. The antibody solution was decanted and the blot was washed four times for 5 min inPBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:15 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 180 seconds.

*Following is the protein extraction buffer composition that was used to extract the total protein from the whole seedlings:50mM Tris, 4% SDS, 1% PVPP , 5% Glycerol, 1mM PMSF, 2mM Pefabloc.

Courtesy of Anshika Jain and Erin Connolly, University of South Carolina, USA