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
low-temperature centrifuge, ice maker, Water bath
chloroform, deionized water
homogenizer (if it is a tissue sample)
reagent preparation
Extraction Buffer
ready-use type; Before use, balance to room temperature. Store at 4 ° C.
ReagentⅠ : ready-to-use; Before use, equilibrate to room temperature; Store at 4 ° C.
ReagentⅡ : ready-to-use; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
ReagentⅢ : ready-to-use; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
standard curve setting: take 20μ L of NaNO2 Standard (10 mmoL/L), using 980 μ L Extraction Buffer diluted to 200 μ mol/L, then further dilute the standards with Extraction Buffer as indicated in the table below.
| NO. | 200 μmol/L NaNO2 Standard volume | ?Extraction Buffer volume | Standard concentration |
|---|
| ?Std.1 | 200 μL | 0 | 200 μmoL/L |
| ?Std.2 | 100 μL | 100 μL | 100 μmoL/L |
| ?Std.3 | 50 μL | 150 μL | 50 μmoL/L |
| ?Std.4 | 20 μL | 180 μL | 20 μmoL/L |
| ?Std.5 | 10 μL | 190 μL | 10 μmoL/L |
| ?Std.6 | 5 μL | 195 μL | 5μmoL/L |
| ?Std.7 | 2 μL | 198 μL | 2 μmoL/L |
| ?Std.8 | 1 μL | 199 μL | 1 μmoL/L |
Sample preparation
1, animal tissues: say 0.1 g sample, add 1 mL Extraction Buffer, ice bath homogenate, 10000 g, 4 ℃ centrifuge for 10 min, take the supernatant, the ice under test.
- Plant tissue: Said to take about 0.1 g sample, add 1 mL Extraction Buffer dolly, ice bath ultrasonic broken 5 min (20% or 200 W, ultrasound 3 s, 7 s interval, repeat 30 times), 10000 g, 4 ℃ centrifuge for 10 min, take the supernatant, ice under test.
- Cells: Collect 5 million cells to the centrifugal tube, with cold PBS cleaning cells, abandoned after centrifugal supernatant, add 1 mL Extraction Buffer, ice bath ultrasonic broken cell 5 min (20% or 200 W, ultrasound 3 s, 7 s interval, repeat 30 times), then 10000 g, 4 ℃ centrifuge for 10 min, take on clear liquid, the ice under test.
4, serum (plasma), such as cell supernatant liquid samples: direct measurement.
Note
if you need to determine protein concentration, it is recommended to use the article number: protein quantitative abs9232 kit (BCA) to determine the protein concentration in the sample.
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.
- Operation table:
| Reagent | control tube (μL) | Blank tube (μL) | Detector tube (μL) | Standard tube (μL) |
|---|
| Different concentrations Std | 0 | 0 | 0 | 40 |
| Sample | 40 | 0 | 40 | 0 |
| Extraction Buffer | 140 | 100 | 60 | 60 |
| ReagentⅠ | 0 | 80 | 80 | 80 |
| The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min |
| Reagent Ⅱ? | 60 | 60 | 60 | 60 |
| Reagent Ⅲ | 60 | 60 | 60 | 60 |
| The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min | The mixture was mixed and incubated in a water bath at 37 ° C for 20 min |
| Trichloromethane | 100 | 100 | 100 | 100 |
| Blending, 8000 g, 25 ℃ for 5 min, learn 200 mu L supernatant on 96 - well plates or trace glass colorimetric determination of melamine in 540 nm absorbance value, remember to A. Determination of Δ A = A - A comparison, Δ A = A standard - A blank. | Blending, 8000 g, 25 ℃ for 5 min, learn 200 mu L supernatant on 96 - well plates or trace glass colorimetric determination of melamine in 540 nm absorbance value, remember to A. Determination of Δ A = A - A comparison, Δ A = A standard - A blank. | Blending, 8000 g, 25 ℃ for 5 min, learn 200 mu L supernatant on 96 - well plates or trace glass colorimetric determination of melamine in 540 nm absorbance value, remember to A. Determination of Δ A = A - A comparison, Δ A = A standard - A blank. | Blending, 8000 g, 25 ℃ for 5 min, learn 200 mu L supernatant on 96 - well plates or trace glass colorimetric determination of melamine in 540 nm absorbance value, remember to A. Determination of Δ A = A - A comparison, Δ A = A standard - A blank. | Blending, 8000 g, 25 ℃ for 5 min, learn 200 mu L supernatant on 96 - well plates or trace glass colorimetric determination of melamine in 540 nm absorbance value, remember to A. Determination of Δ A = A - A comparison, Δ A = A standard - A blank. |
Note
Each sample needs to set a control hole to exclude the influence of NO2- present in the sample itself, so 96 T can only test 48 samples. It is recommended before the experiment to select 2-3 samples with large expected differences for pre-experiment. If ΔA was less than 0.005, the sample size could be increased appropriately. If Δ A greater than 1.0, the samples available Extraction Buffer further dilution, the calculation results multiplied by the dilution ratio, or reduce the Extraction with sample size.
Results calculated
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
Concentration standard solution as the y axis, Δ A standard is x axis, y = kx + drawing standard curve b. Δ A determination to the equation y values (including mol/L).
2, super oxygen anion content calculation
(1) Calculated according to the protein concentration of the sample
Superoxide anion content (μmol/mg prot)=y×V sample ÷(V sample ×Cpr)×10-3=y÷Cpr
Super oxygen anion producing rate (including mol/min/mg prot) = x y V samples present sample (V (Cpr) present T x 10-3 = 0.05 y present Cpr
(2) calculated according to fresh weight of samples
Superoxide anion content (μmol/g fresh weight)=y×V sample ÷(V sample ÷V extract ×W)×10-3=y÷W
Super oxygen anion producing rate (including mol/min/g fresh weight) = x y V samples present present V (V sample extraction x W) present T x 10-3 = 0.05 present W y
(3) volume calculation according to the serum (plasma) or the culture
The ultra oxygen anion content (mu mol/mL) = y x 10-3 = y
The ultra oxygen anion produce rate (including mol/min/mL) = y present T x 10-3 = 0.05 y
Sample volume V samples
participate in response, 0.04 mL; Cpr: sample protein concentration, mg/mL; T: the reaction time, 20 min; V Extraction: the volume of extraction liquid added in the extraction process, 1 mL; W: fresh weight of sample, g; 10-3 mL = 10-3 L.
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