Anti-CUL4 | Cullin-4

Samples (left to right: Col-0, csn5a-1 and csn5b-1)

Total proteins were extracted from the 14 days grown seedlings of Arabidopsis thaliana ecotype Col-0 and mutant csn5a-1 and csn5b-1 (T-DNA mutant SALK_063436 and SALK_007134, respectively) plants using Laemmli SDS sample buffer (2X): 65 mM Tris-HCL (pH 6.8), SDS (3%), glycerol (10%), bromophenol blue (0.005%) and 2-mercaptoethanol (0.5%, v/v). About 50 μg of each extract was denatured by boiling at 75°C for 5 mins. Samples were resolved on 10.0% SDS-PAGE gel and blotted for 3 hr on PVDF membrane using wet transfer at 4°C. The membrane was blocked with 5% skimmed milk for 1 hr at room temperature with mild rocking. The membrane was then incubated in the primary antibody at a dilution of 1:1000 for anti-CUL1 and 1:500 for anti-CUL4 in TBS-T at 4°C overnight with mild rocking. The antibody solution was discarded, and the membrane was washed 3 times for 15 mins each in TBS-T at room temperature with mild rocking. The membrane was then incubated in matching secondary antibody (anti-rabbit IgG HRP-conjugated) diluted to 1:5000 in TBS- T for 1 hr at room temperature with mild rocking. The blot was washed as earlier and developed with the chemiluminescent detection reagent. Exposure time was adjusted to 30 sec.

Courtesy of phd candidate