![]() The technique described may be used with minimal variations to study the effects of stress conditions which lead to glutathione redox changes. Western blot Introduction Keeping the inside of the cell reduced with a high content of protein thiols is a delicate balance between intracellular pro- and antioxidants, essential for cell survival. Complementary to existing redox sensors and conventional redox measurements, nucleus-targeted rxYFP sensors provide a novel tool for examining nuclear redox homeostasis in mammalian cells, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. Glutathionylated Cch1p was detected by western blot analysis with anti-GSH antibody, and significant glutathionylation was observed. The details of Western blotting protocols may vary from applications, with protein characteristic adaptations and the level of information required, however, they all follow some common basic steps. A nucleus-targeted rxYFP sensor can be used to sense nuclear steady-state and dynamic redox changes in response to oxidative stress. Immunoblotting, is a widely used technique for the detection and analysis of proteins. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential ( E) of the Trx1 active site (230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an helix proximal to the active site, which formed u. Examination of thiol/disulfide redox changes in thioredoxin (Trx) family members including Trx1 in cytoplasm and nucleus and Trx2 in mitochondria should aid in the understanding of compartmentalized redox signaling mechanisms. In this chapter we describe methods to measure the nuclear rxYFP redox state in human cells by a redox Western blot technique. Abstract Increasing evidence suggests that compartment - specific changes are important in redox signaling and control. The redox state of TRX-1 was determined in native gels without additon of 2-mercaptoethanol in samples, as described previously 17. Genetically encoded biosensors, including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP), provide an alternative approach to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. Immunoblotting was performed using a standard protocol. Intracellular redox homeostasis is crucial for many cellular functions, but accurate measurements of cellular compartment-specific redox states remain technically challenging. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h.
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