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After oxidant treatment, wash cells with ice-cold phosphate-buffered saline (PBS) and then add 5 mL of PBS containing 40 mM IA for 5 min to prevent post-lysis oxidation of cysteine residues (see Note 2). Aspirate PBS and add 150 pL of digitonin extraction buffer containing freshly added IA to each dish. Scrape and pool cells then transfer to appropriately labeled tubes on ice. Rock tubes on ice for 10 min and centrifuge extraction mixture at 480g for 10 min at 4 C. Transfer supernatant (approx...

Methods

Seed HeLa cells onto 35-mm glass-bottom dishes in D- 2. Allow the cells to grow to 70-80 confluence. 3. Change media for Opti-MEM without serum, 700 pL per dish. Incubate 1 h. Steps 4-7 describe the protocol for one 35-mm glass-bottom dish 4. To a sterile eppendorftube (1.5 ml) add 100 pl serum free Opti-MEM. Add 3 pl of FuGENE 6 transfection reagent (see Note 1). Mix rapidly by vortexing, and allow to stay for 5 min. 5. Add 2 pl (1 pg) of HyPer-Cyto vector. Mix rapidly by...

Matthew Whiteman Yuktee Dogra Paul G Winyard and Jeffrey S Armstrong

Reactive oxygen intermediates (ROIs) play a key role in a number of human diseases either by inducing cell death, cellular proliferation, or by acting as mediators in cellular signaling. Therefore, their measurement in vivo and in cell culture is desirable but technically difficult and often troublesome. To address some of the key methodological issues in examining the formation of ROI in cells and mitochondria, this chapter discusses the following (a) the cellular sources of ROI and their...

Future Directions

Two major limitations of redox-sensitive GFPs remain to be resolved. First, the response time, although notably improved in the re-engineered roGFPs, likely remains too slow to permit accurate indication of transient oxidative bursts involved in some types of signaling events. Second, the highly reducing midpoint potentials of roGFP probes currently limit quantitative applications to reducing compartments such as the cytosol and mitochondria. Strategies to develop roGFP variants with more...

Materials

Purification of Wild-Type p53 Protein 1. Sf9 (Spodoptera frugiperda) insect cells (ATCC, American Type Culture Collection) 2. TNM-FH medium (Grace's medium supplemented with yeastolate and lactalbumin hydrolysate Sigma) + 10 fetal bovine serum (preheat serum at 56 C for 30 min before adding to the medium). 3. NPVp53, a recombinant baculovirus expressing wild-type murine p53 (6) (kindly provided by Peter Tegtmeyer). 4. Lysis buffer 150 mM Tris-HCl, pH 9.0 150 mM NaCl 1 mM ethylene diamine...

Recent Applications of roGFPs

Plant Cell Jiang and co-workers have recently transformed Arabidopsis Expression with roGFP1, targeted to both the mitochondria and cytosol 36 . Intracellularly expressed roGFP1 responded to the addition of exogenous oxidizing and reducing agents in a like manner to that reported for mammalian cells and in vitro assays 20, 22 therefore, the same calibration curves were used to convert percent oxidation of the probe to redox potentials. Parallel monitoring of the redox potential with an...

Andrew G Pinder Stephen C Rogers Afshin Khalatbari Thomas E Ingram and Philip E James

A plethora of publications on techniques and methodologies for measuring nitric oxide NO or reaction products of NO NO metabolites has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on...

Redox GFPs

Section 2 highlights the importance of a reporter protein-based real time redox assay. The advantages of using GFPs and its color variants e.g., yellow fluorescent proteins for a variety of applications and how the chromophore is formed have been reviewed previously see ref. 16, and Chap. 4 . In the context of the use of GFP for redox-sensing in vivo, the opportunity to conduct noninvasive experiments with a protein which is regarded as neutral in its impact on biological systems, contains no...

Mark B Cannon and S James Remington

The quantification of transient redox events within subcellular compartments, such as those involved in certain signal transduction pathways, requires specific probes with high spatial and temporal resolution. Redox-sensitive variants of the green fluorescent protein roGFP have recently been developed that allow the noninvasive monitoring of intracellular thiol-disulfide equilibria. In this chapter, the biophysical properties of these probes are discussed, including recent efforts to enhance...