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Cat. No.: G6PAK-100 (for 100T)

Description

Glucose-6-Phosphate Assay Kit is a high-sensitivity colorimetric assay kit used for detecting G6P content in cells, tissues, or other samples. It utilizes a WST-8-based color reaction for the assay. WST-8 is an upgraded alternative to MTT and has significant advantages over MTT or similar products like XTT and MTS. Firstly, formazan produced by MTT reduction is not water-soluble and requires specific solvents for dissolution, whereas formazan produced by WST-8, XTT, and MTS is water-soluble, eliminating the need for subsequent dissolution steps. Secondly, the formazan produced by WST-8 is more soluble than that produced by XTT and MTS. Thirdly, WST-8 is more stable than XTT and MTS, resulting in more consistent experimental results. Additionally, WST-8 offers a wider linear range and higher sensitivity compared to MTT, XTT, and others. Compared to WST-1, WST-8 provides higher detection sensitivity, easier solubility, and greater stability.

G6P (Glucose-6-phosphate), also known as 6-phosphoglucose, is a molecule formed by the phosphorylation of the hydroxyl group on the sixth carbon of glucose, catalyzed by hexokinase. It is a common small molecule involved in sugar metabolism within cells, participating in biochemical pathways such as glycolysis and the pentose phosphate pathway. In the first step of glycolysis, glucose is catalyzed by hexokinase to form glucose-6-phosphate, which is then converted into fructose-6-phosphate by phosphoglucose isomerase to continue the subsequent steps of glycolysis. In the pentose phosphate pathway, glucose-6-phosphate is the first substrate and serves as the main route for the production of NADPH. In addition to these two metabolic pathways, glucose-6-phosphate can also be converted into glycogen or starch for storage.

Components

• Enzyme Mixture
• G6P Standard (10 mM)
• Color Development Solution
• Reaction Buffer
• G6P Extraction Solution

Principle

Glucose-6-phosphate (G6P) is oxidized by glucose-6-phosphate dehydrogenase (G6PDH) to form 6-phosphogluconate (6-PG), during which NADP+ is reduced to NADPH. The NADPH generated in this reaction reduces WST-8 to produce an orange-yellow formazan in the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate), with a maximum absorption peak around 450 nm. The amount of formazan produced in the reaction system is proportional to the total amount of G6P in the sample.

Features

• This kit is easy to use. It allows detection directly from cell or tissue lysates without the need to isolate and purify G6P from cells, tissues, or other samples.
• The kit offers high sensitivity and a wide linear range. It can detect G6P at concentrations as low as 20 μM (1 nmol) and shows a good linear relationship between 20 μM (1 nmol) and 500 μM (25 nmol).

Storage

Store at -20°C, valid for one year. The Color Development Solution must be stored at -20°C and protected from light.

Precautions

• Tests have shown that storing the enzyme mixture in this kit at room temperature for 72 hours or subjecting it to five freeze-thaw cycles does not affect its enzymatic activity.
• Since the G6P Extraction Solution is slightly viscous, it is important to ensure thorough mixing during dilution, whether diluting the standard or sample. Otherwise, significant fluctuations in experimental data may occur.
• During sample addition and mixing, bubbles should be avoided as much as possible to prevent affecting the final absorbance measurements.
• If it is not possible to strictly control the reaction temperature and time, a standard curve must be set up for each assay.
• If the concentration of G6P in the sample solution is too high or too low and falls outside the linear detection range of the kit, the amount of sample or extraction solution can be adjusted accordingly.
• Whenever possible, use fresh samples for detection, as frozen or repeatedly freeze-thawed cell or tissue samples may affect the results.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment. It must not be used in food or pharmaceuticals or stored in ordinary residences.
• For your safety and health, please wear a lab coat and disposable gloves while handling.