vefhard.blogg.se - Redox western blot technique

Redox western blot technique Activator#

Redox western blot technique Activator#

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Isotope-coded affinity tags (ICAT) analysis.

Here we describe a suite of methods to measure nuclear redox state, which include a redox Western blot technique to quantify the redox state of Trx l, a biotinylated iodoacetamide (BIAM) method for thioredoxin reductase-1 (TrxR1), GSH redox measurement using total protein S-glutathionylation, and a redox isotope-coded affinity tag (ICAT) method for measuring oxidation of specific cysteines in high-abundance nuclear proteins. Redox measurements are obtained under conditions with excess thiol-reactive reagents. The redox states of these systems, including the small redox active protein thioredoxin-1 (Trx1) and the abundant, low molecular weight thiol antioxidant glutathione (GSH), in nuclei provide means to quantify nuclear redox conditions. Uncontrolled oxidation of these thiols causes dysfunction, and two major thiol-dependent antioxidant systems provided protection. These proteins include enzymes, transport machinery, structural proteins, and transcription factors with conserved cysteine in zinc fingers and DNA-binding domains.

Many nuclear proteins contain thiols, which undergo reversible oxidation and are critical for normal function.