Product Specification| Operating Instructions | Serum (plasma), animal and plant tissues, cells, cell supernatant | | 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
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.
2. 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.
3. 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:
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 | 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 | | 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 | | 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. |
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. | | Theory | Superoxide anion (OFR) and other reactive oxygen species (ROS) play important roles in immunity and signal transduction. However, excessive accumulation of ROS can damage cell membranes and biological macromolecules, leading to metabolic abnormalities in cells and tissues, and thus cause a variety of diseases. Superoxide anion in plant, animal tissue, serum and other samples reacts with hydroxylamine hydrochloride to form NO2-, NO2- reacts with Griess reagent, the mechanism of Griess analysis is summarized as azo coupling between diazogens, diazogens are produced by sulfonamide and NO2- and N-(1-naphthyl) ethylenediamine dihydrochloride, resulting in red azo compounds. There is a characteristic absorption peak at 540 nm, and the O2- content in the sample can be calculated according to the A540 value. Samples such as animal and plant tissues, serum and cells can be detected. | | Synonym | Micro Superoxide Anion Assay Kit | | Composition | | Name | Size (96T) | Storage | | Extraction Buffer | 100mL | 4℃ | | ReagentⅠ | 8mL | 4℃ | | Reagent Ⅱ | 6mL | 4℃, keep in dark place | | Reagent Ⅲ | 6mL | 4℃, keep in dark place | | NaNO2 Standard (10 mmoL/L) | 0.5mL | 4℃ |
| | Background | Reactive oxygen species (ROS), such as superoxide anion, play important roles in immunity and signal transduction in organisms. However, excessive accumulation of ROS can damage cell membranes and biological macromolecules, leading to metabolic abnormalities in cells and tissues, which can cause a variety of diseases. | | General Notes | 1. Do not mix the components between different batch numbers and different manufacturers.
2, when mixing or redissolving the components, avoid 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. Own chloroform.
6, each sample needs to set a control hole to exclude the influence of NO2- existing in the sample itself, so 96T can only measure 48 samples. | | Storage Temp. | 2-8℃, stored in the dark, valid for 6 months. | | Applications | Serum (plasma), animal and plant tissues, cells, cell supernatant |
bio-equip.cn AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.
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