PntA (Slr1239) | Pyridine nucleotide transhydrogenase alpha-subunit
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25 µg of total proteins from Synechocystis sp. PCC 6803 (WT) and the ΔpntA mutant extracted with buffer containing 50 mM HEPES-NaOH, pH 7.5, 30 mM CaCl2, 800 mM sorbitol and 1mM 6-aminohexanoid acid and denatured with 2xLaemli buffer at +4 °C for O/N. Proteins were separated on 12 % SDS-PAGE containing 6 M urea and blotted 1h to PVDF using semi-dry transfer. Blot was blocked with 5 % milk for 1h at room temperature (RT) with agitation. After that blot was washed with TTBS for 2 x 5 min and incubated in the primary antibody at a dilution of 1: 3000 in TTBS for O/N at +4 °C with agitation. The antibody solution was decanted and the blot was rinsed briefly with TTBS and washed 4 x 5 min with TTBS at RT with agitation. Blot was incubated in secondary antibody (Amersham ECL Rabbit IgG, HRP-linked whole Ab, from GE Healthcare) diluted to 1:10 000 in for 2h at RT with agitation. The blot was washed 3 x 5 min with TTBS and 2 x 5 min with TBS and developed for 5 min with chemiluminescent detection reagent. Exposure time was around 2 min.
Courtesy of Tuomas Huokko, University of Turku, Finland
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