Methods in Enzymology Vol.353 Redox Cell Biology and Genetics Part B

This chapter describes a novel reagent, biotinylated glutathione ethyl ester (BioGEE), and protocols that allows for the rapid purification of proteins that are oxidatively modified at reactive cysteine thiols in situ. The approach has a number of advantages over existing technologies. Because the tracer molecule can be incorporated into protein only as a consequence of cysteinyl thiol oxidation, the cell can be loaded without the need to inhibit protein synthesis. In addition, excess label is scavenged from the system before and during cell lysis so that incorporation of the label accurately reflects the glutathiolation state of the proteins before cell lysis. Finally, the use of biotin as a label allows sensitive nonradioactive detection and rapid affinity purification of labeled proteins with streptavidin conjugates. By using BioGEE, oxidative modification of several proteins can be demonstrated in conjunction with tumor necrosis factor α (TNF-α)-stimulated apoptosis, and identified two proteins that had not previously been shown to be glutathiolated. The chapter presents detailed protocols for the synthesis, purification, and use of BioGEE.

• Series: Methods in Enzymology (Book 353)
• Hardcover: 634 pages
• Publisher: Academic Press;
  
• Language: English
• ISBN-10: 0121822567
• ISBN-13: 978-0121822569
• Product Dimensions: 6 x 1.4 x 9 inches

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