BC2-tag

Samples:
10 µg A. thaliana – Wt Col-0 (Negative control) (1)
20 µg A. thaliana – Wt Col-0 (Negative control) (2)
10 µg of A. thaliana expressing POI-BC2t fusion (3)
20 µg of A. thaliana expressing POI-BC2t fusion (4)

10 + 20 µg/well of total protein were extracted from Arabidopsis thaliana leaf material in diluted HENS (25mM HEPES pH 7.7, 1mM EDTA, 2.5 % SDS) and stored at -80°C. Samples were denatured in 1x protein loading dye (0.5% Sodium dodecyl Sulfate, 0.002% Bromophenol Blue, 10% glycerol, and 50 mM Tris-HCl pH6.8) at 95°C for 5 min. Samples were separated on 12% SDS-PAGE gel and blotted 1h to a PVDF membrane (pore size of 0.2 µm), using a semi-dry transfer. Blot was blocked with 10% milk in TBS-T o/n at 4°C without agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 in 10% milk in TBS-T, at RT, for 1h with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 in 10% milk in TBS-T at RT for 1h with agitation. The blot was washed as above and developed for 2 min with ECL substrate. Exposure time was 11 minutes and 2 seconds.

Courtesy of Dr. Patrick Treffon, Elizabeth Vierling Lab Department of Biochemistry and Molecular Biology University of Massachusetts Amherst, USA