Description
Reducing sugars (RS) are widely present in animals, plants, microorganisms, and cultured cells. Reducing sugars in plants primarily include glucose, fructose, and maltose. Among these, glucose and fructose are not only the main substrates for respiration but also serve as substrates for the further synthesis of sucrose, starch, and cellulose.Detection Principle: In an alkaline solution, 3,5-dinitrosalicylic acid (DNS) can be reduced by reducing sugars to produce a brown-red-colored amino compound, which has a characteristic absorption peak at 540 nm. Within a certain concentration range, the RS content is linearly correlated with the absorbance at 540 nm. The RS content in the sample can be calculated based on a standard curve.Detection Range: 0.05 - 0.6 mg/mLSensitivity: 0.025 mg/mLApplicable Samples: Plant tissues, animal tissues, cells, bacteria, serum (plasma)R1501790Component48T96TStorageR1501790AExtraction Buffer60 mL120 mL2-8℃R1501790BDNS Reagent10 mL20 mL2-8℃. Store in the dark.R1501790CStandard1EA1EA2-8℃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 visible spectrophotometer (capable of measuring absorbance at 540 nm)96-well plate or micro glass cuvettes, adjustable micropipettes and tipsCentrifuge, water bathDeionized waterHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesExtraction BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C. Slightly irritating. Use appropriate personal protective equipment.DNS ReagentReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light. Slightly irritating. Use appropriate personal protective equipment.StandardBefore use, add 1 mL of deionized water to dissolve, preparing a 10 mg/mL stock standard solution.Can be stored at 4°C for 2 weeks.2. Standard Curve SetupDilute the 10 mg/mL standard stock solution with deionized water to concentrations of 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 mg/mL.TubeVolume of 10 mg/mL Standard (µL)Volume of Deionized Water (µL)Concentration (mg/mL)Std.1609400.6Std.2509500.5Std.3409600.4Std.4309700.3Std.5209800.2Std.6109900.1Std.759950.05Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample Preparation3.1 Plant or Animal Tissue SamplesWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Transfer the homogenate to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.3.2 Bacteria or CellsCollect bacteria or cells into a centrifuge tube; discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Sonicate in an ice bath for 5 minutes (power 20%, pulse 3s on, 10s off, repeat 30 times). Transfer to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.3.3 Serum (Plasma) SamplesTake 0.1 mL of serum (plasma) and add 0.9 mL of Extraction Buffer; mix thoroughly. Transfer to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.Note:If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended. The Extraction Buffer contains components that denature proteins. If calculating based on protein concentration, protein needs to be re-extracted separately for measurement.4. Assay Steps4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 540 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Assay Procedure:ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Control Tube (µL)Sample00175175Standard (various conc.)017500Deionized Water17500125DNS Reagent1251251250Mix well. Heat in a boiling water bath for 5 minutes (cap tightly to prevent evaporation). Remove and immediately cool to room temperature. Transfer 200 µL to a 96-well plate or micro glass cuvette. Measure the absorbance at 540 nm. Calculate ΔA test = A test - A control, ΔA standard = A standard - A blank. Note:The Blank and Standard tubes only need to be set up 1-2 times.It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If ΔA <sub> test </sub> is less than 0.04, consider increasing the sample volume appropriately. If ΔA <sub> test </sub> is greater than the ΔA <sub> standard </sub> of the 0.6 mg/mL standard, further dilute the sample with Extraction Buffer (multiply the result by the dilution factor) or reduce the amount of sample used for extraction.5. Calculation of ResultsNote: We provide both the derived formula and a simplified formula. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.5.1 Standard Curve PlottingPlot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (mg/mL).5.2 Sample Reducing Sugar Content Calculation(1) Based on Sample WeightReducing Sugar (µg/g) = 1000 × y × V<sub>extraction</sub> ÷ W × n = 1000 × y / W × n(2) Based on Sample Protein ConcentrationReducing Sugar (µg/mg prot) =1000 × y × Vextraction ÷ (Vextraction × Cpr) × n=1000 × y / Cpr × n(3) Based on Bacterial or Cell CountReducing Sugar (µg/10⁴) =1000 × y × V<sub>extraction</sub> ÷ 500 × n = 2 × y × n(4) Based on Serum (Plasma) VolumeReducing Sugar (µg/mL) = 1000 × y × Vextraction ÷ Vliquid × n = 10000 × y × nParameter Definitions:1000: Unit conversion factor (1 mg/mL = 1000 µg/mL)V extraction : Volume of Extraction Buffer added (1 mL)V liquid : Volume of serum (plasma) added (0.1 mL)Cpr: Sample protein concentration (mg/mL)W: Sample weight (g)500: Total number of bacteria or cells (5 million)n: Dilution factor6. Representative ResultsTypical Standard Curve: y = 0.2243x + 0.0545, R² = 0.9957 PrecautionsThis product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear lab coats and disposable gloves during operation