application example

1 μg of total protein from three different lines of Arabidopsis thaliana over expressingAtMYC2 (273 aa, 31.4KDa) (2,3,4) and and extract from a mutant line of AtMYC2 where MYC2 band is negligible (1) extracted using a protocol of Mechin et al. 2003, including a coctail of protease inhibitors and separated on 12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 5 % skimmed milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 8 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was 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, from Agrisera, AS09 602) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed with ECL according to the manufacturer's instructions. Exposure time was 7 minutes.

Courtesy of Dr. Sunita Yadav, Indian Institute of Technology Kharagpur, India