application example

Total protein was extracted from WT and a transgenic line of Arabidopsis thaliana expressing a HA-tagged chloroplast protein. The extraction buffer contained 0.2M Tris-HCl pH 6.8, 10% Glycerol, 8% SDS, 20% bME. Proteins were separated on 11% SDS-PAGE, and blotted onto a nitrocellulose membrane. The blot was blocked with 5% non-fat dry milk in 1x TBS-T buffer for 1h at room temperature (RT) with agitation. The blot was incubated with the primary antibody at a dilution of 1: 4 000 for 1h at RT with agitation. The antibody solution was decanted, the blot was rinsed briefly, and washed 3 times (5, 10 and 15 min) in TBS-T at RT with agitation. The blot was then incubated in a secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated; Agrisera, AS09 602, 1:10 000 dilution) for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer instructions. Signal was captured using the GE LAS500 instrument, with exposure time of 30 seconds.

Courtesy of Dr. Zach Adam, The Hebrew University of Jerusalem, Israel