Cell Stress & Metabolism
Applications
- GST activity measurement and quantitation of GST-tagged fusion proteins
- Determination of the concentration of NO metabolites (nitrate/nitrite)
- SOD assays
- HAT activity measurement
- Metabolic studies and analysis of mutagenic, carcinogenic and toxic effects
- DNA damage detection and quantitation
- Analysis of the cell redox state
Benefits
- Highly sensitive and reproducible detection/measurement
- Ready-to-use and convenient assays
- Suited for high-throughput applications
Fluorometric & Colorimetric GST Assay Kits
Glutathione (GSH) is the major intracellular low-molecular-weight thiol and plays a critical role in cellular defense against oxidative stress in tissues and cells. Glutathione S-transferase (GST) is an essential enzyme involved in detoxification of xenobiotics mediating conjugation of glutathione with those compounds.
The Fluorometric and Colorimetric GST Assays can detect GST activity in crude cell lysate or purified protein fractions and the kits also suited for quantitation of GST-tagged fusion proteins. They are based upon the GST-catalyzed reaction between GSH and the GST substrate CDNB (Colorimetric Kit) or MCB (Fluorometric Kit).
CDNB has the broadest range of isozyme detectability (e.g. α-, µ-, phi-, and other GST isoforms). Under certain conditions, the interaction between glutathione and CDNB is totally dependent on the presence of active GST. Detection limit is approximately 10 ng active GST/assay.
The free form of MCB is almost non-fluorescent, whereas the dye fluoresces blue (Ex/Em = 380/461 nm) when reacting with glutathione. GST catalyzes the MCB-glutathione reaction, and the fluorescence levels are proportional to the amount of GST present in the reaction. Therefore, the GST level in samples can be easily detected using a fluorometer or a 96-well fluorometric plate reader.
- Principle of the SOD Assay Kit.
SOD Assay Kit
Superoxide dismutase (SOD) is one of the most important antioxidative enzymes. The highly sensitive SOD Assay Kit utilizes WST-1 that produces a water-soluble formazan dye upon reduction with the superoxide anion. The rate of the reduction with a superoxide anion is linearly related to the xanthine oxidase (XOD) activity, and is inhibited by SOD. Therefore, the inhibition activity of SOD can be determined by a colorimetric method.
Fluorometric & Colorimetric Nitric Oxide Kits
Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune responses, and apoptosis. PromoKine's Fluorometric and Colorimetric Nitric Oxide Kits provide an accurate and convenient tool for total nitrate/nitrite concentration determination in a simple two-step process that can be used as a quantitative measure of NO production. The first step is the conversion of nitrate to nitrite utilizing nitrate reductase. The second step involves the addition of the fluorometric reagent DAN followed by NaOH, which converts nitrite into a fluorescent compound (Fluorometric Nitric Oxide Kits) - or the addition of the Griess Reagent which converts nitrite into a deep purple azo compound (Colorimetric Nitric Oxide Kits). Measurement of the fluorescent compound reaction product or azo chromophore accurately determines the total nitric oxide production. The Fluorometric Assay Kit has been validated in culture media, plasma, and tissue homogenates.
HAT Activity Kit
Histone acetyltransferases (HATs) have been implicated in playing a crucial role in various cellular functions, such as gene transcription, differentiation, and proliferation. The colorimetric HAT Activity Kit offers a convenient, non-radioactive system for a rapid and sensitive, spectrophotometric detection of HAT activity in mammalian samples. The detection can be continuous and is therefore suitable for kinetic studies.
DNA Damage Detection Kit
Different physiological and environmental factors such as chemical induced damage and cell aging are causing DNA lesions. AP sites (apurinic/apyrimidinic sites) are one of the major types of DNA lesions formed during the process of base excision and repair of oxidized, deaminated, or alkylated bases. It has been estimated that about 2x105 base lesions are generated per cell per day. For this reason, the level of AP sites in cells can be a good indicator of DNA lesions and repairs.
The DNA Damage Detection Kit utilizes the ARP (Aldehyde Reactive Probe) reagent that reacts specifically with the aldehyde group in the open ring form of the AP sites. After treating DNA containing AP sites with ARP reagents, AP sites are tagged with biotin residues, which can be quantified using an avidin-biotin assay followed by a colorimetric detection. The kit provides the necessary reagents for convenient determination of abasic sites in purified DNA sample in 96-well plate format.
NAD/NADH & NADP/NADPH Quantitation Kits
PromoKine’s NAD/NADH and NADP/NADPH Quantitation Kits provide a convenient tool for sensitive detection of the intracellular nucleotides NAD, NADH, NADP, and NADPH along with the determination of their ratios. They can be used for studies of energy transformation and the determination of the redox state of cells or tissue. The kits have been designed to specifically recognize NAD/NADH only or NADP/NADPH only in an enzyme cycling reaction that significantly increases detection sensitivity. Purification of NAD/NADH or NADP/NADPH from sample mixes is not required.
Fluorometric & Colorimetric ATP & ADP Quantitation Kits
ATP that is formed exclusively in the mitochondria is the major energy currency of living systems and virtually all energy requiring processes utilize the chemical energy stored in the phosphate bond of ATP. A variety of genetic diseases can affect ATP formation in the mitochondria. There are a number of commercially available ATP assays which detects femtomoles or less of ATP by measuring luminescence (PromoKine Kit PK-CA577-254-200, for example) but these kits require specialized luminescence instrumentation and utilize luciferase which can be difficult to maintain in active form. PromoKine's new Colorimetric & Fluorometric ATP Quantitation Kit is designed to be a robust, simple method which utilizes the phosphorylation of glycerol to generate a product that is easily quantified by colorimetric (λmax = 570 nm) or fluorometric (Ex/Em = 535/587 nm) methods. The assay can detect as low as 50 picomol (1 µM) of ATP in various samples.
ADP is a product of ATP dephosphorylation and it can be rephosphorylated to ATP. De-phosphorylation and rephosphorylation occur via various phosphatases, phosphorylases and kinases. ADP is stored in platelets and can be released to interact with a variety of purinergic receptors. ADP levels regulate several enzymes involved in intermediary metabolism. ADP conversion to ATP primarily occurs within the mitochondrion and chloroplast, although several such processes occur in the cytoplasm. Conventionally, ADP levels are measured by luciferase/luciferin mediated assays after ADP is converted to ATP. However, the luciferase system is unstable and luminescence equipment is not generally available in most laboratories. PromoKine’s new Fluorometric & Colorimetric ADP Quantitation Kit provides an convenient colorimetric and fluorometric means to measure the ADP level. In this assay, ADP is converted to ATP and pyruvate. The generated pyruvate can be quantified colorimetrically (λmax = 570 nm) or fluorometrically (Ex/Em 535/587 nm). The assay is simple, sensitive, stable and high-throughput adaptable and can detect as low as 1 µM ADP in biological samples.
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Note: Oxidative Stress is also topic of a training course of our PromoCell Academy! >More Information