application example

2 µg of Arabidopsis thaliana total protein from 15 days old seedlings (1) from 200ng/μl extracted with buffer containing Tris (pH7.5), NaCl, Triton-x100 and protease inhibitors and yeast protein extracts from SG335 cells transformed with pGADT7/SOC1 construct (2) were separated on 12 % SDS-PAGE and blotted 40 min. to PVDF. Blots were blocked with 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 for 1.5h at RT with agitation. The antibody solution was decanted and the blotwas rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 45min at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 7 min.

Double band in the yeast sample might be due to post-translational mofifications as experiment with yeast harbouring vector showed no cross-reactivity.

Courtesy of Dr Theoni Margaritopoulou, Agricultural University of Athens, Greece