MKK2 | Mitogen-activated protein kinase kinase 2

Application example





Arabidopsis thaliana mesophyll protoplasts (Col-0) were isolated and transformed according to Yoo et al. (2007). 300 µl of protoplasts (2*105 protoplasts/ml) expressing MKK2/MKK4/MKK5-HA (or untransformed protoplasts as control) were pelleted by centrifugation. The resulting pellets were resuspended in standard Laemmli sample buffer and then the samples were denatured at 95°C for 5 min. Samples were separated on 10% SDS-PAGE and blotted 1 h onto NCL membrane using semi-dry transfer. The blot was blocked with 5% skimmed milk in TBS-T for 1h at room temperature (RT) with agitation. The blot was then incubated with the primary antibody (anti-MKK2) at a dilution of 1:1000 for 1 h at RT with agitation in 3% skimmed milk in TBS-T. The antibody solution was decanted and the blot was washed 4 times for 10 min with TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, Agrisera) diluted to 1:15000 in 3% skimmed milk in TBS-T for 1h at RT with agitation. The blot was washed as above and incubated for 5 min with highest sensitivity detection reagent. Arrow indicates endogenous MKK2, star indicates MKK2-HA. Exposure time was 2 minutes. As a control, the blot was re-probed with anti-HA after stripping.

Courtesy Dr. Lennart Eschen-Lippold, Leibniz Institute of Plant Biochemistry, Germany