Anti-CUL1 | Cullin-1

Total proteins were extracted from the fully-expanded leaves of 3-week-old Arabidopsis thaliana ecotype Col-0 and ipk1-1 (T-DNA mutant SALK_065337) plants using a 6 M Urea solution. About 20 μg of each extract was denatured by boiling in 1X Laemmli buffer at 98°C for 10 mins. Samples were resolved on 7.5% SDS-PAGE gel and blotted for 1.5 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 1:5000 in TBS-T at 4°C overnight with mild rocking. The antibody solution was discarded, and the membrane was washed 3 times for 10 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 1:10 000 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 4 sec.

Courtesy of Dr.Saikat Bhattacharjee, Regional Center For Biotechnology, Haryana, India