Chapter title |
Affinity purification and reconstitution of human phagocyte flavocytochrome B for detection of conformational dynamics in the membrane.
|
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Chapter number | 24 |
Book title |
Neutrophil Methods and Protocols
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Published in |
Methods in molecular biology, February 2014
|
DOI | 10.1007/978-1-62703-845-4_24 |
Pubmed ID | |
Book ISBNs |
978-1-62703-844-7, 978-1-62703-845-4
|
Authors |
Riesselman M, Jesaitis AJ, Marcia Riesselman, Algirdas J. Jesaitis, Riesselman, Marcia, Jesaitis, Algirdas J. |
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. |
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