Human flavocytochrome b (Cyt b) is the core electron transferase of the NADPH oxidase in phagocytes and a number of other cell types. The oxidase complex generates superoxide, initiating production of a cascade of reactive oxygen species critical for the killing of infectious agents. Many fundamental questions still remain concerning its structural dynamics and electron transfer mechanisms. In particular, Cyt b structure/function correlates in the membrane have been relatively unstudied. In order to facilitate the direct analysis of Cyt b structural dynamics in the membrane, the following method provides rapid and efficient procedures for the affinity purification of Cyt b from isolated neutrophil membrane fractions and its functional reconstitution in purified lipid preparations. The protocol presented here contains some new optimized procedures that will facilitate Cyt b isolation and reconstitution. Additional methods are presented that facilitate examination of conformational dynamics of the membrane reconstituted purified Cyt b by fluorescence resonance energy transfer (FRET) as measured by steady-state and lifetime fluorescence techniques. © 2014 Springer Science+Business Media, LLC.
CITATION STYLE
Riesselman, M., & Jesaitis, A. J. (2014). Affinity purification and reconstitution of human phagocyte flavocytochrome b for detection of conformational dynamics in the membrane. Methods in Molecular Biology, 1124, 413–426. https://doi.org/10.1007/978-1-62703-845-4_24
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