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Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era

  • Hiroshi Omote
  • Yoshinori Moriyama
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

Abstract

Measuring transport activity through reconstituted proteoliposomes is a key technique to resolve numerous problems found in the traditional methods. The system includes overexpression, purification, and reconstitution of transporters. Mixing of purified transporter with lipid and dilution below the critical micelle concentration result in rapid generation of proteoliposomes. Incubation of proteoliposomes in the presence of a driving force initiates substrate uptake. After starting the reaction, samples are passed through a gel filtration column to separate proteoliposomes from the reaction mixture. Here, we describe step-by-step procedures for such reconstitution assays.

Key words

Transporter Purification Reconstitution Proteoliposome Driving force Kinetics 

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Membrane BiochemistryOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityOkayamaJapan

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