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. Author manuscript; available in PMC: 2016 Feb 29.
Published in final edited form as: Methods Enzymol. 2014 Dec 26;552:185–210. doi: 10.1016/bs.mie.2014.10.007

Figure 8.

Figure 8

Peroxiredoxin oxidation cycles in fruit flies. Two independently generated polyclonal antisera against over-oxidised peroxiredoxin (PRX-SO2/3) demonstrate circadian pattern in the over-oxidation of PRX in head lysates of Drosophila melanogaster, sampled under constant dark (DD) conditions (the 2nd and 3rd DD cycles are shown). The custom-made antiserum was generated by Cambridge Research Biochemical (CRB) using a ‘double purification’ approach: The crude serum was first depleted of non-sulfonylated epitopes using a peptide containing serine (–OH) in its (i.e. DFTFVSPTEI) followed by retrieval of antigen-specific antibodies against the antigen peptide (sulfonylated sequence, DFTFV[C-SO3]PTEI). The membranes were re-probed with anti-2-Cys PRX antibody (1:1,000 dilution) as a loading control. Abcam ab16830 and CRB anti-PRX-SO2/3 were used at 1:10, 000 and 1:1,000 dilution, respectively. Marker used: MagicMark XP (Life Technologies).