| Usage | Self-supplied consumables:
microplate reader or visible spectrophotometer (can measure the absorbance at 540 nm)
96-well plate or microglass cuvette, adjustable pipet gun and tip
centrifuge, Water bath
deionized water
homogenizer (if it is a tissue sample)
Reagent preparation:
Extraction Buffer: ready-use type; Before use, equilibrate to room temperature; Store at 4 ° C.
DNS Reagent: ready-to-use; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
Standard: Add 1 mL deionized water to dissolve, prepare 10 mg/mL standard before use; Store at 4 ° C.
standard curve setting: 10 mg/mL standard was diluted to 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 mg/mL with deionized water.
| NO. | 10 mg/mL Standard volume（µL） | Deionized water volume（µL） | Concentration（mg/mL） | | Std.1 | 60 | 940 | 0.6 | | Std.2 | 50 | 950 | 0.5 | | Std.3 | 40 | 960 | 0.4 | | Std.4 | 30 | 970 | 0.3 | | Std.5 | 20 | 980 | 0.2 | | Std.6 | 10 | 990 | 0.1 | | Std.7 | 5 | 995 | 0.05 |
Sample preparation:
1. Plant or animal tissue samples: About 0.1g of tissue was weighed, 1mL of Extraction Buffer was added, homogenized in an ice bath, and transferred to a covered centrifuge tube (to prevent water evaporation during heating). The mixture was shaken and mixed every 5 min in a water bath at 80 ° C for 40 min, centrifuged at 8000 g at 25 ° C for 10 min, and the supernatant was taken for measurement.
2, bacteria or cells: collecting bacteria or cells to the centrifugal tube, abandon the supernatant; Add 1 mL Extraction Buffer per 5 million bacteria or cells, break the bacteria or cells by ultrasound in an ice bath for 5 min (20% power, 3S ultrasound, 10S interval, repeat 30 times), transfer to a covered centrifuge tube (to prevent water evaporation during heating), and water bath at 80℃ for 40 min. The mixture was shaken and mixed every 5 min, centrifuged at 8000 g for 10 min at 25 ° C, and the supernatant was taken for measurement.
3, serum (plasma) samples: 0.1 mL serum (plasma), plus 0.9 mL Extraction Buffer, fully blending; Transfer to a covered centrifuge tube (to prevent water evaporation during heating), water bath at 80 ° C for 40 min, shake and mix once every 5 min, centrifuge at 8000 g for 10 min at 25 ° C, and take the supernatant to be measured.
Note: To measure protein concentration, it is recommended to use abs9232 Protein Quantification kit (BCA assay).
Experimental procedures:
1. The microplate reader or visible spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 540 nm. The visible spectrophotometer was zeroed with deionized water.
2. Operation table:
| Reagent | Blank tube（µL） | Standard tube（µL） | Detector tube（µL） | Control tube（µL） | | Sample | 0 | 0 | 175 | 175 | | Different concentration Std. | 0 | 175 | 0 | 0 | | Deionized water | 175 | 0 | 0 | 125 | | DNS Reagent | 125 | 125 | 125 | 0 | Mix well, heat in a boiling water bath for 5 min (cover tightly to prevent evaporation of water), remove and immediately cool to room temperature. Take 200 mu to 96 L orifice or trace glass dishes, and 540 nm wavelength determination of absorbance values. ΔA assay =A assay − A control and ΔA standard =A standard − A blank were calculated |
The results were calculated as follows:
Note: We provide you with the calculation formula, including the derivation process calculation formula and the concise calculation formula. The two are exactly the same. The concise formula in bold is recommended as the final formula.
1. Drawing of the standard curve
The standard curve was plotted with the concentration of the standard on the Y-axis and the ΔA standard on the X-axis. Bringing Δ A determination to the equation to calculate the y value.
2, sample reducing sugar content calculation
(1) according to the sample quality is calculated
Reducing sugar (μg/g)= 1000 ×y×V extraction ÷W×n= 1000 ×y÷W×n
(2) calculated according to the protein concentration of the sample
Reducing sugar (mu/mg prot g) = 1000 * y * V extraction present (V extraction (Cpr) * n * n = 1000 * y present Cpr
(3) According to the number of bacteria or cells
Reducing sugar (μg/104)= 1000 ×y×V extraction ÷500×n=2×y×n
(4) according to the serum (plasma) volume calculation
Reducing sugar (mu g/mL) = 1000 extraction liquid present V * y * V * y * n = 10000 n
1000: unit conversion factor, 1 mg/mL= 1000 μg/mL; V extraction: to join
Extraction Buffer volume, 1 mL; V solution: Add serum (plasma) volume, 0.1 mL;
Cpr: sample protein concentration, mg/mL; W: sample quality, g; 500: total number of bacteria or cells, 5 million; n: dilution factor |
| Theory | Reducing sugars (RS) are widely found in animals, plants, microorganisms and cultured cells. Reducing sugars in plants mainly include glucose, fructose and maltose, among which glucose and fructose are not only the main substrates for respiration, but also the substrates for further synthesis of sucrose, starch and cellulose. The reducing sugar content detection kit (micromethod) can detect animal and plant tissues, bacteria, cells and serum (plasma) and other liquid samples. The principle is that in alkaline solution, 3, 5-dinitrosalicylic acid can be reduced by RS to produce brown red amino compounds, with a characteristic absorption peak at 540 nm, within a certain concentration range. There is a linear relationship between the RS content and the absorbance at 540 nm. According to the standard curve, the RS content in the sample can be calculated. |
| General Notes | 1. Do not mix the components between different batch numbers and different manufacturers; Otherwise, it may lead to abnormal results.
2, when mixing or redissolving the components, avoid air bubbles.
3, frequently change the suction head to avoid cross contamination between the components.
4, before the experiment, ensure that all the components and equipment are at the right temperature.
5. Before the experiment, it is recommended to select 2-3 samples with large expected differences for pre-experiment. If the absorbance value of the sample is not within the measurement range of the standard curve, it is recommended to dilute. 6. DNS Reagent has certain toxicity, please take protective measures during operation. |
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