PEPCK | PEP carboxy kinase

application example

20 µg of total protein from (1) Arabidopsis thaliana total cell extracted with Protein Extration Buffer, PEB (AS08 300), (2) Phaseolus vulgaris total cell, extracted with PEB, (3) Zea mays total cell extracted with PEB, (4) Miscanthus giganteus total cell extracted with PEB, (5) Panicum virgatum total cell extracted with PEB, (6) Spartina alterniflora  total cell extracted with PEB, (7)  Spartina patens  total cell extracted with PEB, were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Abcam) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).