Anti-CLO1 | Caleosin-1

Total protein from Arabidopsis thaliana mature seeds were extracted with extraction buffer 100 mm Tris-HCl, pH 8.5, 4% [w/v] SDS, 2% [v/v] β-mercapthoethanol, and 2 mm phenylmethylsulfonyl fluoride and separated on 12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 5% non-fat milk in TBST for 1 h at room temperaturę with agitation. Membranes were incubated in the primary antibody at a dilution 1:1000 in 1% non-fat milk in TBST at 4°C overnight. Then, membranes were washed 3 Times for 15 min in TBST at RT with agitation. Membranes were incubated in secondary antibody (Anti-rabbit IgG horse radish peroxidase conjugated from Agrisera AS09 602) diluted to 1: 50 000 in 1% non-fat milk in TBST for 1h at RT with agitation. The blots were washed as above and developed for 5 min with ECL (Agrisera ECL Bright AS16 ECL-N) according to the manufacturer's instructions.

Courtesy Dr. Agnieszka Zienkiewicz, Dept. of Plant Physiology and Molecular Biology of Plants, Nicolaus Copernicus University, Toruń, Poland