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Affinity Purification and Reconstitution of Human Phagocyte Flavocytochrome b for Detection of Conformational Dynamics in the Membrane

  • Marcia Riesselman
  • Algirdas J. Jesaitis
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

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.

Keywords

Phagocyte Reactive oxygen species Flavocytochrome b Immunoaffinity purification Reconstitution Membrane conformational dynamics Fluorescence lifetime analysis 

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Marcia Riesselman
    • 1
  • Algirdas J. Jesaitis
    • 1
  1. 1.Department of MicrobiologyMontana State UniversityBozemanUSA

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