application example

Total proteins (containing 2.5 to 30 ug ) from Chlamydomonas cells 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 10 % 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. Blot was incubated in the primary antibody (∆TMCrPDAT) at a dilution of 1:250 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, from Bio-Rad) 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 (Bio-Rad) 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 of Dr. Kangsup Yoon, Arizona State University.