| Description | Glutathione S-Transferase (GST) is a protein family with multiple physiological functions, primarily located within the cytoplasm. GST is a crucial component of the body's detoxification enzyme system. It mainly catalyzes the covalent binding of the sulfhydryl group of glutathione (GSH) to Glutathione S-Transferase (GST) is a protein family with multiple physiological functions, primarily located within the cytoplasm. GST is a crucial component of the body's detoxification enzyme system. It mainly catalyzes the covalent binding of the sulfhydryl group of glutathione (GSH) to various chemicals and their metabolites, converting electrophilic compounds into hydrophilic compounds that are more easily excreted in bile or urine. This process facilitates the degradation and elimination of potentially toxic substances from the body. Thus, GST plays a vital biological role in protecting cells from damage caused by electrophilic compounds. GST possesses GSH peroxidase activity (also known as non-Se-GSH-Px) and functions in repairing oxidatively damaged macromolecules such as DNA and proteins. The GST-catalyzed reaction consumes GSH but does not increase GSSG levels. Detection Principle: GST catalyzes the conjugation of GSH with CDNB (1-chloro-2,4-dinitrobenzene). The conjugation product has an absorption peak at 340 nm. The GST activity is calculated by measuring the rate of increase in absorbance at 340 nm. Detection Range: 2 - 76 U/L Sensitivity: 2 U/L Applicable Samples: Serum (plasma), animal/plant tissues, cells, bacteriaG1501773Component48T96TStorageG1501773AAssay Buffer60 mL120 mL2-8℃G1501773BChromogen11 mL22 mL2-8℃. Store in the dark.G1501773CSubstrate1EA1EA2-8℃. Store in the dark.Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.User-Prepared Instruments and ReagentsMicroplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)96-well UV plate or micro quartz cuvettes, adjustable micropipettes and tipsIce maker, refrigerated centrifuge, water bathDeionized waterHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesAssay BufferReady-to-use; Equilibrate to room temperature before use.Store at 4℃.ChromogenReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light. Skin irritant. Use appropriate protective equipment.Substrate Working ReagentBefore use, dissolve in 2.4 mL of deionized water.Unused reagent can be stored at 4°C protected from light for one month.2. Sample Preparation2.1 Animal/Plant TissuesWeigh 0.1 g of tissue sample. Add 1 mL of pre-cooled Assay Buffer and homogenize quickly on ice. Centrifuge the homogenate at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.2 Cells or BacteriaCollect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Assay Buffer. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.3 Serum (Plasma)Assay directly.Note:Sample processing should be performed on ice. If not used immediately, samples can be stored at -80°C for one month.For GST activity measurement in cells, the cell count should be between 3-5×10⁶. Use Assay Buffer with sonication for cell extraction; do not use cell lysis buffers.If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.3. Assay Steps3.1 Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. For UV spectrophotometers, zero the instrument with deionized water.3.2 Incubate the Substrate Working Reagent at 25°C (for general species) or 37°C (for mammals) for 15 minutes.3.3 Add reagents to a 96-well UV plate or micro quartz cuvette as follows:ReagentBlank (µL)Test (µL)Sample020Assay Buffer200Chromogen180180Substrate Working Reagent20203.4 Mix rapidly and immediately measure the change in absorbance at 340 nm. Record the absorbance for the Blank at 10 seconds (A1) and 310 seconds (A2). Record the absorbance for the Test at 10 seconds (A3) and 310 seconds (A4). Calculate ΔA blank = A2 - A1, ΔA test = A4 - A3. Note: Only one Blank is needed. A preliminary test with 2-3 samples showing expected significant differences is recommended. If the sample absorbance is greater than 1, dilute the sample with deionized water and multiply the result by the dilution factor. The reaction temperature significantly affects the results; maintain it at 25°C (general species) or 37°C (mammals). 4. Calculation of Results 4. Calculation of Results Note: We provide both derived and simplified calculation formulas. They are equivalent. The simplified formulas in bold are recommended for final calculation. 4.1 Calculation Formulas for 96-well UV Plate (1) Based on Protein Concentration Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 µmol of CDNB with GSH per minute per milligram of protein at 25°C or 37°C. Calculation Formula: GST Activity (U/mg prot) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (Cpr × Vsample) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ Cpr (2) Based on Sample Fresh Weight Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 µmol of CDNB with GSH per minute per gram of sample at 25°C or 37°C. Calculation Formula: GST Activity (U/g fresh weight) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (Vsample ÷ Vtotal sample × W) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ W (3) Based on Cell or Bacterial Count Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 µmol of CDNB with GSH per minute per 10⁴ cells or bacteria at 25°C or 37°C. Calculation Formula: GST Activity (U/10⁴) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (500 × Vsample ÷ Vtotal sample) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ 500 (4) Based on Liquid Volume Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 µmol of CDNB with GSH per minute per milliliter of liquid at 25°C or 37°C. Calculation Formula: GST Activity (U/mL) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ Vsample ÷ T = 0.46 × (ΔAtest - ΔAblank) 4.2 Calculation Formulas for Micro Quartz Cuvette Adjust the pathlength (d) in the formulas above from 0.5 cm to 1 cm for calculations. Parameter Definitions: ε: Molar extinction coefficient of the product, 9.6 × 10³ L/mol/cm d: Light path for 96-well plate (0.5 cm) 10⁶: Conversion factor (1 mol = 1 × 10⁶ µmol) V total reaction : Total reaction volume (220 µL = 2.2 × 10⁻⁴ L) Cpr: Protein concentration of the supernatant (mg/mL) W: Sample weight (g) V sample : Volume of supernatant added to the reaction system (20 µL = 0.02 mL) V total sample : Volume of extraction buffer added (1 mL) T: Reaction time (5 min) 500: Cell or bacterial count factor (5 × 10⁶ total cells / 10⁴ per unit = 500)Precautions1. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.2. This product is for research use only. Not for use in clinical diagnosis... 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