CTR1 | Constitutive triple response 1

30 µg/well of total protein was extracted from frozen leaves  with 100 mM Tris-HCl buffer pH 7.6 (containing 12.5% Glycerol, 20 mM β-mercapthoethanol, 2 mM PMSF) and denatured with Sample Buffer at 70°C for 5 min. The proteins were separated on 10 % SDS-PAGE and blotted 1.5 h to nitrocellulose membrane (pore size of 0.45 µm), using wet transfer. Blot was blocked with 5 % milk for 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in TBS-T containing 2% milk ON/4°C 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 in TBS-T for 1.5 h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECLBright (AS16 ECL-N-10). Exposure time was 10 seconds.

Courtesy of Dr.Irina Vaseva,Laboratory 'Regulation of Gene Expression', Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria