application information

The Arabidopsis thaliana wild-type (WS) and ahk2-2T-DNA insertion mutant plants were grown on MS/2 media for six days.Tissue was collected, frozen in liquid nitrogen, ground in 3 volumes of 2x SDS-PAGE loading buffer and heated at 95 C for 5 min. After 10 min centrifugation at RT, 20 µl of the supernatant was loaded per lane on a 7.5% TGX gel (BioRad). After separation, proteins were blotted 1 hr onto a supported nitrocellulose membrane.Blots were blocked with 10% milk in PBS-T for 15 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody diluted 1: 10, 000 in 1% milk in PBS-T for 16h at 4C with agitation. The antibody solution was decanted and the blot was washed 3 times for1 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 2000 in PBS-Tfor 1h at RT with agitation. The blot was washed as above and developed for 5 min withSuperSignal WestFemtoSubstrateaccording to the manufacturers instructions. Chemiluminescent signal was captured using ChemiDoc equipped with a CCD camera.

Courtesy of Dr. Jasmina Kurepa, University of Kentucky College of Agriculture, Food and Environment, USA