Biotin carboxyl carrier subunit of chloroplast ACCase
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Purified native protein from Pisum sativum
Hordeum vulgare, Nicotiana tabacum
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20 µg of a total cell extract leaves of Nicotiana tabacum (1), Arabidopsis thaliana (2), Solanum lycopersicum (3), Nicotiana tabacum (4), Hordeum vulgare (5) were separated on 12 % SDS-PAGE and blotted 1h to nitrocellulose using tank transfer. Blots were blocked with 5 % milk in TBS for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 for 1h at RT 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 RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 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 15 seconds.
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