DGAT2A | Acyl-CoA: Diacylglycerol acyltransferase

kDa

application example

Total proteins (containing 30 ug ) from Chlamydomonas reinhardtii cells grown for the indicated times in N-deprived medium extracted with lysis buffer (50 mM Tris-HCl, pH 6.8, containing 2% SDS and 10 mM EDTA and a protease inhibitor cocktail) were separated on 12 % SDS-PAGE and transferred onto a nitrocellose blot over night at 4°C. Blots were blocked with blocking buffer ( 5% (w/v) non-fat dry milk powder in TBS-T) for 2 hrs at room temperature (RT) with agitation. Blots were incubated in the primary antibody (CrTMDGAT2A) was used at a dilution of 1:1000 over night at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then whashed 5 times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:5000 in the same buffer for 1h at RT with agitation. The blot was washed as above and developed for 5 min with Chemiluminescence detection kit according to the manufacturers instructions. An imaging system (ChemiDoc XRS; Bio-Rad) was used to quantitatively and qualitatively analyze protein blot. Exposure time was 30 seconds.

Courtesy Dr. Yantao Li, The University of Maryland, USA