Anti-SQS1 | Squalene synthase

60 µL of tissue extracted freshly from Arabidopsis thaliana seedlings was ground with a pestle and three volumes of sample buffer [(500 mM Tris pH 8.0, 4% (w/v) lithium dodecyl sulfate, 1 mM EDTA, 20% (w/v) glycerol, 11 μM Coomassie blue G250, 16.6 μM phenol red, 50 μM dithiothreitol (DTT)] was added. Proteins were denatured by boiling the samples at 100°C for 5 min. Samples were separated at room temperature on 10% SDS-PAGE (Bolt Bis-Tris gels) and transferred (wet) for 15 minutes to nitrocellulose (pore size 0.45 μm), using an eBlot L1 transfer system (GenScript). Blot was blocked with 5% BSA for 1 h at room temp with rocking. Blot was incubated in the primary antibody at a dilution of 1:500 overnight at 4°C with rocking. The antibody solution was decanted, and the blot was rinsed briefly, then washed 3 times for 5 min in TBS-T at room temp with rocking. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:2 500 in 5% BSA for 1 hour at room temp with rocking. The blot was washed as above and developed with WesternSure Premium Chemiluminescent substrate (Thermo Fisher, 50-489-552) for 3 minutes. Exposure time was 3 minutes.

Courtesy of Dr. Bonnie Bartel, Rice University, USA