application example

10 µg of total protein from 7 day old Arabidopsis plants grown at control conditions, 22°C wilde type, Columbia-9 (1), hot1-3, HSP101 null (2), clpBp-1, ClpBp null (3) and heat shocked at 38°C for 1.5 hour wilde type, Columbia-9 (4), hot1-3, HSP101 null (5), clpBp-1, ClpBp null (6) were separated on 7-16 % SDS-PAGE and blotted overnight to nitrocellulose membrane. Blots were blocked immediately following transfer in for 1h at room temperature with agitation. Blots were incubated in Agrisera anti-ClpB-P antibody (AS09 459) at a dilution of 1: 1000 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 GE healthcare) diluted to 1:2500 in 5% dry milk blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent according to the manufacturers instructions. Exposure time was 20 seconds.

Courtesy Minsoo Kim, University of Arizona