Highly Specific Affinity Purified Polyclonal Antibodies
Circadian rhythms affect many metabolic pathways, for example the phylogenetically conserved peroxiredoxin (PRX) proteins, show daily rhythms in oxidation state. To explore the biochemical properties of the PRX system, Dr John O’Neill, based at the Laboratory of Molecular Biology in Cambridge enlisted the assistance of Cambridge Research Biochemicals to prepare a custom made high affinity rabbit polyclonal anti-PRXSO2/3 antibody. The polyclonal anti-sera demonstrates a clear circadian rhythm in the over-oxidation of PRX in head lysates of Drosophila melanogaster sampled under constant dark conditions.
Right: Peroxiredoxin oxidation cycles in fruit flies. Two independently generated polyclonal antisera against over-oxidized peroxiredoxin demonstrate circadian pattern in the overoxidation of PRX in head lysates of Drosophila melanogaster.
Above: This was sampled under constant dark (DD) conditions (the 2nd and 3rd DD cycles are shown). The custom-made antiserum was generated by Cambridge Research Biochemicals using a “double purification” approach: The crude serum was first depleted of non-sulfonylated epitopes using a peptide containing serine followed by retrieval of antigen-specific antibodies against the antigen peptide (sulfonylated sequence, DFTFV[C-SO3]PTEI).
Publication reference: O’NEILL, J et al, Analysis of the Redox Oscillations in the Circadian Clockwork, Methods in Enzymology, 2015, 552, 185-210
CRB offer over 37 years of expertise in the generation of target specific polyclonal antibodies
DR JOHN O’NEILL – MRC LMB
“In many different model organisms and cell types, we had observed a circadian rhythm in the oxidation state of the highly conserved 2-cys peroxiredoxin family of antioxidants. As we investigated the underlying mechanism however, we found we were hindered by variations in the specificity of commercial available antisera against this oxidative protein modification. CRB generated a highly specific affinity-purified polyclonal, additionally depleted for activity against unmodified peroxiredoxin.”
“This tool allowed us to further validate our original findings and obviated any need to take batch variation into account, thereby increasing experimental reproducibility. CRB’s service was efficient and professional, and very much tailored towards our particular needs. I do not hesitate to recommend them to colleagues.”