Anti-CBP60G | Calmodulin-binding protein 60 G

WT is Col-0 and mutant is cbp60g/sard1 double mutant. 1/2LS plate-grown 2-week-old Arabidopsis whole seedlings were harvested and snap frozen in liquid N2. Total protein was extracted using RIPA buffer (see below). Protein concentration was measured using Bradford assay and normalized to 40ug/sample. Equal volume of 2x LPS buffer (containing 10% 2-ME) was added to denatured protein sample. Denatured at 95C for 10 minutes. Samples were separated on 4-12% SDS-PAGE and blotted for 1h to PVDF (pore size of 0.45 um) using iBlot semi-dry transfer. Blot was blocked with 5 % non-fat milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5,000 in 5 % milk ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly once, then washed the blot with PBS-T (PBS with 0.05% tween-20) at RT for 10 min with agitation. Blot was incubated in Goat anti-rabbit ( Agrisera AS09 602 lot 2007)diluted to 1: 20,000 with 5% non-fat milk for 1h/RT with agitation . The blot was washed with PBS-T 3 . Thermo Scientific SuperSignal West Dura Maximum Sensitivity Substrate.
RIPA buffer: 50mM Tris-HCl, pH 7.5, 150 mM NaCl 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, 0.5 mM EGTA

Courtesy of Dr. Yu Ti Cheng, Duke University, USA