application example

10 ul of leaf extract which was equivalent to 1 mg leaf material was loaded per lane, which may also correspond to approximately 50 ug protein. Arabidopsis thaliana seedlings were grown on vermiculite for 3 weeks under continuous illumination at a light intensity of 80 uE m-2 s-1 at 22°C. Twenty mg leaf material was collected from mature leaves and extracted with 200 ul of the tissue homogenization buffer. 10 ul of leaf extract which was equivalent to 1 mg leaf material was loaded per lane. Detection Protocol: Leaf protein was separated on 14% SDS-PAGE and blotted 2h to PVDF membrane from GE Healthcare. The blot was blocked with PBS-T (PBS plus 0.1% tween 20) containing 3% skim milk for 1h at room temperature (RT: approximately 22 degrees C) with gentle agitation. The blots were briefly washed twice with PBS-T and then incubated with anti-GluTR antibody which was diluted 1:1000 with PBS-T for 1h at RT with agitation. The primary antibody solution was decanted and the blot was rinsed twice, when washed once for 10 min and 3 times for 5 min in PBS-Tcontaining 0.5% (w/v) skim milk at RT with agitation. The blot was incubated with the secondary antibody (HRP-conjugated anti-rabbit IgG) which was diluted 1:20 000 with PBS-T containing 0.5% (w/v) skim milk for 1h at RT with agitation. The blot was washed as described above and incubated with Western Lightning Plus-ECL from Perkin-Elmer for