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
- 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
- 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 | Mix well, bath in boiling water for 10 min (cover tightly to prevent water loss), remove and cool to room temperature | Mix well, bath in boiling water for 10 min (cover tightly to prevent water loss), remove and cool to room temperature | 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 | 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 | 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 | 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
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