application example

From 0.1-1.2 µg of chlorophyll from Arabidopsis thaliana total leaf extract, extracted with buffer A (330 mM sorbitol, 25 mM Tricine pH 7.8, 1 mM EDTA, 10 mM KCl, 0,15 % BSA, 4 mM Na ascorbat (L-ascorbic acid), 7 mM L-cysteine) were separated on 15 % SDS-PAGE gel and blotted 1h to PVDF. Blots were blocked with 10% skimmed non fat milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1 000 over night at 4ºC with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera) diluted to 1:10 000 in TTBS for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer’s instructions. Exposure time was 1-3 min.

Courtesy of Dr. Rikard Fristedt, UCLA, USA.