RPS14 | Ribosomal protein S14 (mitochondrial)
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30 µg of mitochondrial fraction from Arabidopsis thaliana extracted with buffer (sucrose, tetrasodiumpyrophosphate, EDTA, KH2O4, PVP-40, BSA, pH 7.5) and denatured with 56°C for 15 min at 650 rpm were separated on 12 % SDS-PAGE and blotted overnight (12V) to PVDF using wet transfer. Blots were blocked with 5% skimmed powder milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the purified anti S14 serum at a dilution of 1: 1 000 for 1,5h 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, from Agrisera, AS09 602) diluted to 1:25 000 in for 1h at RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent. Exposure time was ~ 60 seconds.
Courtesy of Dr. Małgorzata Kwaśniak-Owczarek, University of Wrocław, Poland
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