HYL1 | Hyponastic leave phenotype ds-RNA binding protein

Application example


40 µg of total protein from Arabidopsis thaliana rosette leaves extracted with extraction buffer (100 mM Tris HCl, pH 7.5; 10% glycerol; 5 mM EDTA; 5 mM EGTA; 150 mM NaCl; 0.75% Triton X-100; 0.05% SDS; 1 mM DTT; 1x Complete Mini EDTA-free protease inhibitor (Roche)) were separated on 12 % SDS-PAGE using semi-dry transfer and blotted 1h to PVDF. Blots were blocked with 5% milk in TBS-T O/N at 4°C with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1,5 h at RT with agitation. The antibody solution was decanted and the blot was washed 3 times for 10 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 1:50 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescence detection reagent, according to the manufacturer's instructions. Exposure time was 60 seconds.  There were no other bands present on this blot in applied conditions

Courtesy of Dr. Dorota Raczyńska, M.Sc Agata Stepień Adam Mickiewicz University, Poland