CLO3 | Caleosin-3
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Arabidopsis thaliana senescent leaves were freshly extracted with 2x SDS-sample buffer (+ 2ME) for SDS-PAGE (crude extract) and denatured with 4X SDS buffer at 95°C for 5 min. 10 µg of protein was loaded/well and were separated on 15-20 % SDS-PAGE and blotted 1h to PVDF membrane. Blot was blocked with 3 % skim milk/TBS-T, 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 in TBS-T for 1h/RT. The antibody solution was decanted and the blot was washed 4 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h/RT with agitation. The blot was washed as above and developed with a chemiluminescent detection reagent, following manufacture's recommendations.
Shimada et al. (2010). A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 2010 Feb 1;61(3):519-28. doi: 10.1111/j.1365-313X.2009.04060.x
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