SOT12 | Cytosolic Sulfotransferase 12

Immunoblotting of Arabidopsis thaliana total protein extracts with anti-SOT12 antibody (αSOT12). In the image above wild-type Col-0 is presented next to the SOT12-over-expressing mutant rcd1. The fragment of this image has been published in [https://elifesciences.org/articles/43284], Fig 6B. Leaves from mature plants were harvested and snap-frozen in liquid nitrogen. Frozen leaf material was ground to fine powder. Lysis buffer was added that contained 2% SDS, 20 mM Tris-HCl (pH 7.8), and x 100 protease inhibitor cocktail (Sigma-Aldrich #P9599). Upon addition of the buffer, the samples were vigorously mixed and then incubated 20 min at +37 ̊C. Supernatant was cleared by centrifugation for 5 min at room temperature on the desk-top mini-centrifuge at full speed. Protein concentration was measured using DC Protein Assay Kit II (Bio-Rad #5000112). Samples were equilibrated on equal protein basis. 100 μg of total protein was loaded per well. Protein was separated in precast 4-15 % gradient mini-gels (Bio-Rad #4561085DC) and transferred to PVDF membrane by wet transfer overnight at +4 ̊C and 130 mA, in 1 x Laemmli buffer supplemented with 20 % methanol. The membrane with transferred protein was blocked for 1 hour in TBS/Tween-20 (0.01 %) with 3 % BSA. αSOT12 antibody (diluted x 1 500 in 3 % BSA/TBS/Tween) was added, followed by overnight incubation at +4 ̊C. The membrane was washed 6 x 5 minutes with TBS/Tween. Secondary anti-rabbit HRP-conjugated antibody was added, diluted x 5 000 in 3 % BSA/TBS/Tween, and the membrane was incubated for 1 hour at room temperature. The membrane was washed 6 x 5 minutes with TBS/Tween. Immunoblotting signal was detected by chemiluminescence using the chemiluminescent detection reagent and the UVP BioSpectrum imaging system. The exposure time was 2 min.

Courtesy of Dr. Alexey Shapiguzov, Helsinki University, Finland