TOC1-P | TIMING OF CAB EXPRESSION 1 Arabidopsis clock gene, phosphorylated

8 µg/well of total protein extracted freshly from 10-day-old Arabidopsis thaliana seedlings with extraction buffer (100 mM Tris·Cl, pH 7.5/150 mM NaCl/0.5% NP-40/1mM EDTA/3mM DTT/1 mM PMSF/2 mM NaF/2mM Na3VO4/1 µg ml-1 leupeptin/1 µg ml-1 aprotinin/1 µg ml-1 pepstatin/5 µg ml-1 antipain/5 µg ml-1 chymostatin/50 µM MG132/50 µM MG115/50 µM ALLN) and denatured at room temperature 24 °C for 3 min (TOC1 protein is very labile and sensitive to heat). Proteins were separated on 8% SDS-PAGE regular gels and blotted 1.5h to PVDF (pore size of 0.2 µm), using wet transfer. Blot was blocked with 5% milk for 1.5h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in 5% non-fat milk TBS-T ON/4°C. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 10 min in 5% non-fat milk TBS-T at RT with agitation. Blot was incubated in Agrisera goat anti-rabbit IgG (AS09 602) diluted to 1:25 000 in 5% non-fat milk for 1h/RT. The blot was washed 3 times for 10 min in TBS-T at RT with agitation and developed for 5 min with AgriseraSuperBright (AS16 ECL-S-10). The band marked by a lower arrow is an endogenous TOC1. 

Courtesy of Dr. David E. Somers, Department of Molecular Genetics, The Ohio State University, USA