Methods

Grow Sf9 insect cells at 25 C in TNM-FH medium sup- Wild-Type p53 Protein plemented with 10 fetal calf serum until the cells reach 60-70 confluency in 100-mm plates. Remove the medium, and to each plate add 1.6 mL of a 1 1 dilution of high-titer NPVp53 recombinant baculovirus in TNM-FH medium plus 10 fetal calf serum. Rock the plates for 20 min at room temperature to allow for infection of the cells by the virus, and then add 9 mL of TNM-FH medium plus 10 fetal calf...

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...

Info

Various experiments were designed to test this. Seeds of etr1-1 mutant plants were obtained from the stock center. Simultaneously, the corresponding wild type background plant seeds were also obtained. 3.6.2. Phenotypic Analysis of Plants 1. When the seeds have been obtained, store them at 4 C for at least 48 h, to break the dormancy of the seeds. 2. Sow both wild type and mutant 4 tr1-1 mutant seeds in separate trays of Levington's F2 compost with sand, after wetting the compost until it forms...