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Apoptosis

Volume 559 of the series Methods in Molecular Biology pp 205-217

Date:

Detection of Uncoupling Protein-2 (UCP2) As a Mitochondrial Modulator of Apoptosis

  • Zoltan DerdakAffiliated withDivision of Gastroenterology & Liver Research Center, Brown Medical School and Rhode Island Hospital
  • , Tamako A. GarciaAffiliated withDivision of Gastroenterology & Liver Research Center, Brown Medical School and Rhode Island Hospital
  • , Gyorgy BaffyAffiliated withDivision of Gastroenterology & Liver Research Center, Brown Medical School and Rhode Island HospitalBrigham and Women’s Hospital, Harvard Medical School and VA Boston Healthcare System

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Summary

There is an increasing evidence that uncoupling protein-2 (UCP2), a recently identified molecular sensor and suppressor of mitochondrial reactive oxygen species (ROS), plays an important role in ­regulating apoptosis in different cell systems. A great technical difficulty that many groups have encountered is the reliable detection of endogenously or exogenously expressed UCP2 protein. The goal of this ­chapter is to introduce the reader to techniques that we have successfully used over the years to detect UCP2 protein in various mouse and human specimens. These techniques include mitochondrial isolation and submitochondrial fractionation followed by Western blotting and UCP2 immunohistochemistry. We find that sample preparation is a key to success and it allows one to produce relevant and important data using commercially available antibodies.

Key words

UCP2 Mitochondria Oxidative stress Superoxide Mitochondrial isolation Submitochondrial fractionation Digitonin Alkali treatment Immunohistochemistry Apoptosis