Oxidative stress, which leads to DNA damage, is a primary driver leading to the accumulation of mutations which occurs in living organisms. The estimated frequency of oxidatively-induced lesions is approximately 104/cell/day. The most extensively investigated, and most abundant DNA lesion generated following exposure to hydroxyl radicals, is 8-hydroxy-2’-deoxy-guanosine (8-oxo-dG). Trevigen now offers two products to enable investigators to monitor changes in 8-oxo-dG levels: a high throughput (HT) 8-oxo-dG ELISA Kit, and 8-oxo-dG monoclonal antibody for immunohistochemical, and immunocytochemical detection. Trevigen also offers research kits targeting biomarkers of oxidative stress. These kits employ well-established methods to assay for superoxide dismutase (SOD), glutathione reductase, glutathione peroxidase, or glutathione, which directly and indirectly measure cellular defensive responses against oxidative stress. Cellular responses to oxidative stress are of interest to investigators of a wide variety of human diseases such as: cancer, diabetes, atherosclerosis, stroke, Alzheimer’s, many auto-immune diseases, and aging.
8-Oxo-2'-deoxyguanosine (8-oxo-dG), a frequently used biomarker of oxidative DNA damage, is removed from DNA by the base excision repair pathway, and subsequently transported into saliva, urine and plasma. In order to measure the total pool of 8-oxo-dG, Trevigen has developed a validated competitive ELISA kit that quantifies 8-oxo-dG in DNA, plasma, urine and saliva in the ELISA Kit II. Trevigen’s HT 8-oxo-dG ELISA kit II, employs a 96 strip wells pre coated with 8-oxo-dG, an anti-8-oxo-dG monoclonal mouse antibody, an HRP conjugated secondary antibody, and colorimetric detection substrate to construct a high throughput assay flexible for your experimental design. The 8-OHdG monoclonal antibody binds competitively to 8-oxo-dG immobilized on pre-coated wells and in solution. Antibody bound to 8-oxo-dG in the sample is washed away while antibody bound to 8-oxo-dG attached to the well is retained. Detection of the retained antibody is performed using an HRP conjugate and colorimetric substrate. Product formation is inversely proportional to the amount of 8-oxo-dG present in the sample.
Researchers looking to quantify 8-oxo-dG in DNA will prefer the second generation kit to the first, as the first generation does not quantify 8-oxo-dG in DNA. Both kits come in colormetric format and offer a robust assay flexible to individual research.
Superoxide Dismutases (SOD) catalyze the dismutation of the superoxide radical (O2 -) into hydrogen peroxide (H2O2) and elemental oxygen (O2), and as such, provides an important defense against the toxicity of superoxide radical. SOD1 and SOD2-deficient mice spontaneously develop liver cancer and are prone to develop tumors, whereas over expression of SOD protects tumor cells from apoptosis.
In Trevigen’s Superoxide Dismutase Assay, ions generated from the conversion of xanthine to uric acid, and hydrogen peroxide by xanthine oxidase (XOD), convert NBT to NBT-diformazan. NBT-diformazan absorbs light at 550 nm. SODs reduce superoxide ion concentrations and thereby lower the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity present in experimental samples. The assay is free of interference by other catalytic activities, and is ideal for assaying SOD in mammalian cell lysates. The kit contains the proper lysis buffer and the reagents needed for 100 experimental tests, 50 positive controls, and 50 negative controls. Unlike some other assay kits for SOD, this system is not greatly disturbed by trace metals. Each assay requires only about five minutes, and after a simple calculation, the percent inhibition of the formation of NBT-diformazan by SOD is converted to the relative activity of the sample.
Trevigen’s Glutathione Reductase Assay Kit is a higher volume assay designed to measure cellular levels of Glutathione Reductase, an enzyme which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) (figure1). GSH is an important intracellular antioxidant that reacts with oxygen free radicals and organic peroxides and acts as a substrate for other detoxification enzymes such as glutathione peroxidase and glutathione S-transferase. The activity of glutathione reductase is an important measure of the antioxidant status of the cell. Glutathione reductase requires NADPH for its activity, resulting in the reduction of GSSG to GSH and the corresponding oxidation of NADPH to NADP+.
Trevigen’s Glutathione Assay utilizes a carefully optimized enzymatic recycling method, which employs Glutathione Reductase, for the quantification of glutathione. The sulfhydryl group of glutathione reacts with DTNB (5,5’-dithiobis-2-nitrobenzoic acid, Ellman’s reagent) and produces a yellow-colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB) that is concomitantly produced, is reduced by Glutathione Reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to the concentration of GSH in the sample. Thus, measurement of the absorbance of TNB at 405 or 412 nm provides an accurate estimation of the amount of glutathione in the sample.
Glutathione peroxidase (GP) is found in mammalian cells and helps to prevent lipid peroxidation of cell membranes by consuming free peroxide in the cell. Thus, Glutathione Peroxidase plays a critical role in protecting the cell from free radical damage. Tetrameric Glutathione Peroxidase catalyzes the reduction of H2O2 to water, and organic peroxides to the corresponding stable alcohols, by using glutathione as a source of reducing equivalents. It requires selenium as a cofactor and contains a selenocysteine amino acid residue in the active site of each monomer that participates in the actual mechanism of the enzyme. Glutathione Reductase (GR), provided with each kit, then reduces the oxidized glutathione to complete the cycle. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the absorbance at 340 nm is directly proportional to the Glutathione Peroxidase activity in the sample. Trevigen’s Glutathione Peroxidase Assay Kit can be used to measure glutathione-dependent peroxidases in plasma, erythrocyte lysates, tissue homogenates, and cell lysates.
This high throughput glutathione reductase assay kit is designed to measure cellular levels of Glutathione Reductase, an enzyme which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) (Figure 1). GSH is an important intracellular antioxidant that reacts with oxygen free radicals and organic peroxides and acts as a substrate for other detoxification enzymes such as glutathione peroxidase and glutathione S-transferase. The activity of glutathione reductase is an important measure of the antioxidant status of the cell. Glutathione reductase requires NADPH for its activity, resulting in the reduction of GSSG to GSH and the corresponding oxidation of NADPH to NADP+.
Trevigen's Glutathione Reductase Assay Kit is a spectrophotometric assay in which the oxidation of NADPH to NADP+ is monitored by the decrease in absorbance at 340 nm. This rate of decrease in absorbance at 340 nm is directly proportional to the glutathione reductase activity in the sample because the enzyme is present at rate limiting concentrations. This kit provides the user with all the reagents and plates to easily and rapidly assay for glutathione reductase in cell and tissue extracts.