application example

10 µg of total protein from Zea mays roots (1), Phaseolus vulgaris leaves (2) or roots (3) extracted with a mixture of 250 mM sorbitol, 50 mM Tris–HCl (pH 8), 2 mM EDTA, and protease inhibitors [1 mM phenylmethylsulfonyl Xuoride, 1 mg ml^-1 each of leupeptin, aprotinin, antipain, chymostatin, and pepstatin were separated on 12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 5% milk in TBS-T for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3.000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed four times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:30 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 60 seconds.

Courtesy of Dr. Ricardo Aroca, CSIC, Spain