| Description | The carbon nutritional status in plants and the quality characteristics of agricultural products are often evaluated using sugar content as an important indicator. Monosaccharides and some oligosaccharides (such as maltose) contain free aldehyde or ketone groups, possess reducibility, and are The carbon nutritional status in plants and the quality characteristics of agricultural products are often evaluated using sugar content as an important indicator. Monosaccharides and some oligosaccharides (such as maltose) contain free aldehyde or ketone groups, possess reducibility, and are classified as reducing sugars. Polysaccharides and sucrose are non-reducing sugars. The total sugar content can be determined by measuring the monosaccharide content after hydrolysis, utilizing the property that non-reducing sugars can be hydrolyzed to monosaccharides by acid.Detection Principle: Reducing sugars are oxidized to sugar acids under alkaline heating conditions, while 3,5-dinitrosalicylic acid (DNS) is reduced to a brownish-red amino compound. Within a certain range, the amount of reducing sugar is proportional to the color intensity of the brownish-red product. The absorbance of this brownish-red substance is measured at 540 nm using a microplate reader. This absorbance value has a linear relationship with the reducing sugar content. The reducing sugar and total sugar content in the sample are calculated using a standard curve.This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes.P1501777Component100T300TStorageP1501777AGlu Standard (1 mg/mL)1 mL1 mL2-8℃P1501777BDNS Detection Solution10 mL30 mLRT. Store in the dark.P1501777CColor Solution (for Total Sugar)5 mL10 mLRT. Store in the dark.User-Prepared Instruments and Reagents1. Distilled water, Hydrochloric acid solution, Sodium hydroxide solution2. 50 mL centrifuge tubes, 1 mL centrifuge tubes, Centrifuge, Water bath or incubator, Microplate reader, 96-well plate, Water bathExperimental Procedure1. Extraction of Reducing Sugars1.1 Weigh 0.5-3 g of plant sample, cut into pieces, add about 3 mL of distilled water and homogenize. Transfer to a beaker or conical flask. Rinse the grinder 2-3 times with 12 mL of distilled water and transfer the rinsate to the same container.1.2 Incubate in a 50°C water bath for 30 min, stirring occasionally to ensure thorough extraction of reducing sugars.1.3 Transfer the precipitate and extract to a 50 mL centrifuge tube. Centrifuge at 4000 g for 5 min.1.4 Collect the supernatant. Add 20 mL of distilled water to the precipitate, mix well, and centrifuge again at 4000 g for 5 min.1.5 Collect the supernatant. Combine the supernatants from the two steps. Dilute to 100 mL with distilled water (this is the extract). Mix well. This serves as the test solution for reducing sugars.2. Hydrolysis and Extraction of Total Sugars2.1 Weigh 0.5-3 g of plant sample, cut into pieces, add about 3 mL of distilled water and homogenize. Transfer to a beaker or conical flask. Rinse the grinder 2-3 times with 12 mL of distilled water and transfer the rinsate to the same container.2.2 Add 10 mL of 6 M hydrochloric acid solution to the container, mix well, then heat in a boiling water bath for 30 min for hydrolysis, stirring occasionally.2.3 Take 2 drops and place on a glass slide, add 1 drop of Color Solution (about 50 µL) to check if hydrolysis is complete. If hydrolysis is complete, no blue color should develop.2.4 After hydrolysis, cool to room temperature. Add 6 M sodium hydroxide solution to adjust the pH to 7.4. Dilute to 100 mL with distilled water, mix well. Centrifuge at 4000 g for 5 min or filter.2.5 Take 10 mL of the supernatant or filtrate and dilute to 100 mL with distilled water, creating a 10-fold diluted total sugar hydrolysate (extract). Take 50 µL of this total sugar hydrolysate to measure its reducing sugar content.3. Glucose Standard PreparationTake clean centrifuge tubes or test tubes and prepare a series of Glu standards according to the table below.Standard Working SolutionGlu Standard (1 mg/mL) (mL)Distilled Water (mL)Concentration (mg/mL)10.010.040.220.020.030.430.030.020.640.040.010.850.0501.04. Assay SetupTake 1 mL centrifuge tubes. Set up Blank, Standard, and Test wells according to the table below. Add solutions sequentially, avoiding bubbles. Mix carefully. If the sugar concentration in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up 2-3 replicate wells for samples and take the average.Reagent (µL)Blank WellStandard WellTest WellDistilled Water50//Glu Standard (1-5)/50/Extract//50DNS Detection Solution100100100Heat accurately in a boiling water bath for 5 min. Remove, cool to room temperature with tap water. Add 250 µL distilled water.5. Reducing Sugar MeasurementMix well. Transfer 300 µL sequentially to the corresponding wells of a 96-well plate. Measure the absorbance of Standard and Test wells at 540 nm, using the Blank well to zero the instrument.6. Result Calculation6.1 Standard Curve PlottingUsing the Glu standards (1-5), i.e., the standard glucose concentrations (mg/mL) as the x-axis and the corresponding absorbance values as the y-axis, plot the standard curve. Find the corresponding glucose concentration on the standard curve based on the absorbance of the extract.6.2 Content CalculationPercentage Content of Reducing Sugars:Reducing sugar content per 100 g sample (g) = (c × V T ) / (m × 1000) × 100 = (c × V T ) / (m × 10)Percentage Content of Total Sugars:Total sugar content per 100 g sample (g) = (c × N × V T ) / (m × 1000) × 100 × 0.9 = (c × N × V T ) / (m × 10) × 0.9Parameter Descriptionc: Sugar amount found from the standard curve (mg/mL)V T : Total volume of the extract, 100 mLm: Mass of the plant sample, gN: Dilution factor of the total sugar hydrolysate, 10Precautions1. Avoid repeated freeze-thaw cycles for the aforementioned low-temperature reagents to prevent inactivation or decreased efficiency.2. If test samples cannot be assayed immediately, store at 2-8°C; stable for 3 days.3. If the sample reducing sugar concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.4. The total sugar calculation formula is used when there are few interfering impurities and the reducing sugar content is relatively small compared to the total sugar content. Multiplying by 0.9 accounts for the water consumed during the hydrolysis of total sugars to monosaccharides.5. 6 M Hydrochloric Acid Preparation: Generally, commercially available concentrated hydrochloric acid is 11.6-12 M. Mix concentrated hydrochloric acid with distilled or deionized water 1:1 (v/v) to prepare 6 M HCl. Caution: Hydrochloric acid dissolution in water releases heat; handle carefully to avoid injury.6. 6 M Sodium Hydroxide Preparation: Dissolve 24 g of sodium hydroxide in distilled or deionized water, make up to 100 mL. Caution: Sodium hydroxide dissolution in water releases heat; handle carefully to avoid injury.7. Use reagents promptly after opening to avoid affecting subsequent experimental results... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Inquire | This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. Purified lactose antibodies were coated on a microplate to produce solid-phase antibodies. Lactose was added to the microplate of the coated monoclonal antibody, along with HRP labeled lactose antigens, to compete for binding. After thorough washing, the substrate TMB was added for colorimetry. The depth of sample color is negatively correlated with the content of lactose in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the content of lactose in the sample through a standard curve.Kit composition:130times concentrated washing solution20ml×1 bottle8.1Standard S1（80µg/L）0.5ml×1bottle2Enzyme-linked immunosorbent assay6ml×1 bottle8.2Standard S2（40µg/L）0.5ml×1bottle3Enzyme labeling coated plate96 holes x 1 pieces8.3Standard S3（20µg/L）0.5ml×1bottle4Color reagent A solution6ml×1 bottle8.4Standard S4（10µg/L）0.5ml×1bottle5Color developer B solution6ml×1 bottle8.5Standard S5（5µg/L）0.5ml×1bottle6Stop solution6ml×1 bottle9Instructions1 copy7Sample Diluent6ml×1 bottle10Microplate Sealers2 sheetsSpecimen requirements:1. Specimen processing:(1) After collecting the water sample, it is repeatedly freeze-thawed three times at -20 ℃, and then filtered through glass fiber for future reference(2) The tissue samples should be extracted using butanol: methanol: water (5:25:70 V: V: V), or extracted according to relevant literature. The experiment should be conducted as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃ for future reference2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).Operation steps:1. Sample addition: Set up standard wells, blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the other steps are the same), and sample wells to be tested. Add 50 microliters to the standard well on the enzyme-linked immunosorbent assay (ELISA) plate, and first add 40 diluents to the sample well to be tested µ l. Then add 10 more samples to be tested µ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.2. Enzyme addition: Add 50 enzyme labeled reagents to each well µ l. Excluding blank holes.3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 60 minutes.4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.6. Color development: Add color development agent A50 to each well first µ l. Add color developer B50 again µ l. Gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes7. Termination: Add 50% termination fluid to each hole µ l. Terminate the reaction (at this point, the blue color immediately turns yellow).8. Measurement: Zero the blank hole and sequentially measure the absorbance (OD value) of each hole at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.Calculation:Draw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.Notes:1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple (x n x 5).5. The sealing film is only for one-time use to avoid cross contamination.6. Please store the substrate in dark.7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader8. All samples, washing liquids, and various waste should be treated as infectious substances.9. The components of this reagent with different batch numbers shall not be mixed.Detection range:two µ G/L-90 µ G/L... Read More | Inquire |