Anti-DGAT1 | O-acyltransferase

25 µg/well of total protein was extracted freshly from pennycress (Thlaspi arvense) seeds using the following extraction buffer: 0.1 M Tris-HCl, ph 7.5 containing: 20% glicerol; 1mm EDTA; 10 mM MgCl2; 14 mM B-Mercaptoethanol; Pefabloc 100 ug/mL; 1 ug/ml leupeptin; 1 ug/ml antipain and denatured with (x4) Tris-HCl (ph 6,8), SDS 8%, Glicerol 10%; Bromophenol blue 0.08 %; DTT 80 mM at 95°C/5 10 min. Samples were separated in the cold on 12.5 % SDS-PAGE containing 4M urea, and blotted for 1 h to PVDF membrane, using wet transfer in the cold. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 1h/RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 in for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: Supersignal west-femto (Thermo Fischer Scientific). Exposure time was 25-35 seconds.

Note: to decrease background use protein load/well of 5-10 µg/well and primary antibody at 1: 2500 1h/RT incubation.

Courtesy of Dr. Miguel Alfonso, EEAD-CSIC, Spain