| 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:
Hydrochloric Acid: ready to use; Before use, equilibrate to room temperature; Store at 4 ° C.
Sodium Hydroxide: ready-to-use type; Before use, balance 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: dilute 10mg/mL standard with deionized water to 2, 1.6, 1, 0.8, 0.4, 0.2, 0.1 mg/mL | NO. | 10 mg/mL Standard volume (µL) | Deionized water volume (µL) | Concentration (mg/mL) | | Std.1 | 40 | 160 | 2 | | Std.2 | 32 | 168 | 1.6 | | Std.3 | 20 | 180 | 1 | | Std.4 | 16 | 184 | 0.8 | | Std.5 | 8 | 192 | 0.4 | | Std.6 | 4 | 196 | 0.2 | | Std.7 | 2 | 198 | 0.1 |
Sample preparation:
1. Plant or animal tissue samples: Said to take about 0.1 g group, add 1 mL Hydrochloric Acid, 1.5 mL deionized water, homogenate, boiling water bath for 30 min, add 1 mL of Sodium Hydroxide, blending, deionized water constant volume to 10 mL, 8000 g, 25 ℃ centrifuge for 10 min, take on the solution under test.
2, serum (plasma) samples: Take 0.1 mL serum (plasma), add 0.1 mL of Hydrochloric Acid, 0.15 mL deionized water, homogenate, boiling water bath for 30 min, add 0.1 mL Sodium Hydroxide blending, deionized water constant volume to 1 mL, 8000 g, 25 ℃ centrifuge for 10 min, take on the solution under test.
Note: Protein quantification kit abs9232 (BCA assay) is recommended for protein concentration determination.
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, in the EP tube, in turn, to join the following reagents:
| Reagent | Blank tube（µL） | Standard tube（µL） | Detector tube（µL） | | Sample | 0 | 0 | 30 | | Different Concentration Std. | 0 | 30 | 0 | | Deionized water | 30 | 0 | 0 | | DNS Reagent | 30 | 30 | 30 | | Mix well, bath in boiling water for 10 min (cover tightly to prevent water loss), remove and cool to room temperature | | Deionized water | 180 | 180 | 180 | | Blending, take 200 mu to 96 L orifice or trace glass dishes, and determine the hole of absorbance at 540 nm, respectively for A blank, A standard and A determination, determination of calculation Δ A = A - A blank, Δ A = A standard - A blank |
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. Will be Δ A measurement into the formula to calculate the y value (mg/mL).
2, sample calculated total sugar content
(1) according to the sample quality is calculated
Total sugar (mg/g)=(y×V sample)÷(W×V sample ÷ total V sample)×n=10×y÷W×n
(2) calculated according to the protein concentration of the sample
Total sugar (mg/mg prot) = (x y V sample) present sample (V (Cpr) * n n = y present Cpr
(3) according to the serum (plasma) volume calculation
Total sugar (mg/mL)=y×V sample ÷(V liquid ×V sample ÷V sample total)×n=10×y×n
V-like: added sample volume, 0.03 mL; The volume V sample total: tissue samples, 10 mL; Cpr: sample protein concentration, mg/mL; W: sample quality, g; V solution: liquid sample volume, 0.1 mL; The volume V sample total: liquid samples, 1 mL; n: dilution factor |
| Theory | Carbohydrate is one of the important components of animals and plants, and is also the main raw material and storage material for metabolism. "Total sugars mainly refer to glucose, fructose, lactose, which are reduced, and sucrose, maltose, which can be hydrolyzed to reduced monosaccharides under the measured conditions, and starch, which may be partially hydrolyzed." The total sugar content detection kit (micromethod) can detect the total sugar content in liquid samples such as animal and plant tissues and serum (pulp). Its principle is to hydrolyse the total sugar into reducing sugar, which reduces DNS to amino compounds after co-heating with DNS reagent under alkaline conditions. In a certain concentration range, the reducing sugar content is linear with the absorbance at 540 nm. According to the standard curve, the total sugar content in the sample can be calculated. |
| Background | Total sugars mainly refer to glucose, fructose, lactose, which are reduced, and sucrose, maltose, which can be hydrolyzed to reduced monosaccharides under the measured conditions, and starch, which may be partially hydrolyzed. Carbohydrate is one of the important components of animals and plants, and is also the main raw material and storage material for metabolism. |
| 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 or increase the sample size for detection, and multiply the result by the dilution.
6, Hydrochloric Acid and Sodium Hydroxide are corrosive, DNS Reagent has certain toxicity, please take protective measures during operation.
7, this kit for cellulose decomposition degree can not reach 100%. |
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