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Methods for Determining the Modification of Protein Thiols by Reactive Lipids

Elsevier Science & Technology
DOI: 10.1016/s0091-679x(06)80021-x
  • Part Iv
  • Oxidative Stress Measurements
  • Biology


Publisher Summary This chapter describes the techniques involved in the preparation, characterization, and quantitation of biotin-tagged lipids, as well as the use of biotin reagents to analyze thiols in biological samples. There are four major advantages of biotin—the high affinity of avidin and streptavidin for biotin makes this a very sensitive and specific way to detect the tag; the binding of streptavidin, unlike an antibody, is not readily affected by flanking residues at the site of protein modification; affinity resins are available for the purification of lipid–protein adducts; and the biotin tag can be quantitated easily and accurately using biotinylated standards. One of the most versatile techniques is to attach biotin covalently to thiol-reactive molecules and label thiol-containing proteins. After separation by proteomics methods, the biotin tag can be detected at a level of sensitivity in the picomole range, using Western blot analysis and horseradish peroxidase-coupled streptavidin. The chapter presents two protocols for labeling. The high-pH protocol will deprotonate and allow biotinylated iodoacetamide (BIAM) to label most free protein thiols. This is useful for global analysis and screening of a wide population of thiol containing proteins. The low-pH protocol is useful for analyzing the redox state of a subset of highly reactive protein thiols. Due to their low pKa, they will remain deprotonated and susceptible to biotin tagging at low pH. The chapter also describes biotin-based methods for the quantitation of protein thiols and adduct formation with electrophilic lipids. These methods can be used in a variety of applications, including the assessment of modification of an individual protein.

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