Malondialdehyde (MDA) Assay Kit(for Cell samples) servicebio G4300-48T
$50.00
In stock
SKU
G4300-48T
Product Information
| Product Name: Malondialdehyde (MDA) Assay Kit (for Cell samples) | Cat. No.: G4300-48T |
| Spec.: 48 T | G4300-96T |
| 96 T |
Product Description/Introduction
Free radicals are the intermediate metabolites of various biochemical reactions in human life activities, which can attack polyunsaturated fatty acids in biofilms and trigger lipid peroxidation. Lipid peroxides are formed in the process of lipid peroxidation, among which malondialdehyde (MDA) is one of the common peroxidation products of organisms.Therefore, MDA level is often used to evaluate the level of lipid peroxidation in cells or tissues, and is applied to the research fields such as oxidative stress and iron death.
Based on the principle that MDA can react with thiobarbituric acid (TBA) to form a browny-red complex under high temperature environment, and can be detected by absorbance or fluorescence intensity, this kit can effectively detect MDA quantitatively in cell samples after optimization and debugging by our R&D team. In addition, antioxidants are provided in this kit, which can avoid additional oxidation of the sample during the detection process, making the detection result more accurate.
Storage and Shipping Conditions
Ship with wet ice; Anti-oxidant stored at -20℃ away from light; Other components are stored at 2-8℃ away from light for 6 months.
Product Content
| Component Number: G4300-1 | Component: MDA standard (200 μM) |
| G4300-48T: 1 mL | G4300-96T: 1 mL |
| Component Number: G4300-2 | Component: MDA Detection Probes |
| G4300-48T: 1 (dry powder) | G4300-96T: 2 (dry powder) |
| Component Number: G4300-3 | Component: MDA Assay Buffer |
| G4300-48T: 10 mL | G4300-96T: 20 mL |
| Component Number: G4300-4 | Component: Antioxidant reagents |
| G4300-48T: 200 μL | G4300-96T: 2×200 μL |
Assay Protocol / Procedures
1. Cell Sample Preparation: After cell collection, cells can be resuspended using a suitable buffer or IP lysate, approximately 100-200 μL of reagent per 1 × 10^7 cells, and lysed by sonication or on ice; after treatment, centrifuge at 4°C, 10,000 g for 10-15 min, and remove the supernatant for the subsequent MDA assay;
2. Test Preparation:
2.1. Preparation of MDA probe working solution: The MDA detection probe is fully dissolved by boiling 5 mL of ultrapure water; and add 5 mL of glacial acetic acid (self-provided) in the same volume as the ultrapure water to mix well, and then formulate into the working solution of the MDA probe (can be stored for one month at 4℃);
2.2. MDA test working solution preparation: according to the number of samples measured (including standards), refer to the following table for reasonable preparation;
| Component: MDA Assay Buffer | 1 sample: 200 μL |
| 10 samples: 2000 μL | 50 samples: 10 mL |
| Component: MDA Probe Working Solution | 1 sample: 200 μL |
| 10 samples: 2000 μL | 50 samples: 10 mL |
| Component: Antioxidant reagents | 1 sample: 4 μL |
| 10 samples: 40 μL | 50 samples: 200 μL |
2.3. Preparation of MDA standard: Use PBS or ultrapure water to dilute the MDA standard (200 μM) in gradient (e.g. 50/25/12.5/6.25/3.125 μmol/L for absorbance method; 10/8/6/4/2 μmol/L for fluorescence intensity method) as required, and use it for the data analysis of the Standard Curve Method after the follow-up assay with the samples to be tested; Or appropriately diluted into a known concentration of the standard, with the sample to be tested for subsequent detection, for standard conversion method data analysis;
3. MDA Detection:
3.1. Mix the sample and the MDA assay working solution thoroughly according to the table below;
| standard tube | blank tube |
| sample tube | MDA standard |
| 20 μL | PBS or water |
| 20 μL | Samples to be tested |
| 20 μL | MDA assay working solution |
| 400 μL | 400 μL |
| 400 μL |
3.2. Incubate at 95°C for 40 min;
3.3. Immediately after the incubation, an ice bath was applied for 5 min;
3.4. After the ice bath, centrifuge 10000 g for 10 min, take 200 μL supernatant into a transparent 96-well plate, and use an enzyme marker to detect the absorbance at 532 nm.Or 200 μL in black 96-well plate was used to detect fluorescence intensity (Ex 525/Em 553).It is recommended that the absorbance method is preferred for detection. When the sample concentration is too low, for example, below 1 μmol/L, the fluorescence intensity method can be used for detection.
4. Data analysis:
Before data analysis, the protein concentration of the tissue or cell samples to be tested needs to be calculated. The protein concentration of the sample to be tested can be determined with the BCA Protein Quantitative Detection Kit (G2026).
4.1. Standardized Curve Method:
4.1.1. Draw a standard curve based on the gradient-diluted standard group value-blank group value data: Y=a X+ b (Y is the standard group value-blank group value; X is the MDA concentration of the standard; a is the slope of the standard curve; and b is the intercept of the standard curve);
4.1.2. MDA concentration calculation of cell samples:
Unit sample MDA (μmol/gprot)=[(sample group value-blank group value)-b]÷a×sample dilution÷sample protein concentration (gprot/L)
4.2. Standard product conversion method, MDA concentration calculation of cell samples:
Unit sample MDA (μmol/gprot)=(sample group value-blank group value)÷(standard group value-blank group value)×standard MDA concentration (μmol/L)×sample dilution÷sample protein concentration (gprot/L)
Note
1. Gently shake the reagents before use to ensure that the components are homogeneous.
2. When heating in a water bath, take care to avoid spilling the liquid by boiling.
3. This kit can be used for MDA detection by absorbance and fluorescence intensity method, when the concentration of the sample is too low, it is recommended to use the fluorescence intensity method for detection; if the equipment does not allow it, it can be used by increasing the proportion of the sample in the mixture of the sample and the MDA detection work solution; it can also be used to appropriately prolong the incubation time of 95 ℃.
4. When analyzing the data by the standard conversion method, the concentration of the sample to be tested and the standard should be within the linear detection range of the kit; when analyzing the data by the standard curve method, make sure that the R2 of the standard curve is more than 0.99.
5. For your health and safety, please wear lab coat and gloves during operation.
Appendix 1: Technical parameters
| Testing Indicators | MDA concentration |
| Sample type | Cell samples |
| Absorbance Detection Sensitivity and Detection Range | 0.548 μmol/L; 0.548-100 μmol/L |
| Fluorescence Intensity Method Detection Sensitivity and Detection Range | 0.036 μmol/L; 0.036-10 μmol/L |
| Recovery rate | 101.3% |
| Intra-batch CV | 2.97% |
| Inter-batch CV | 5.06% |
Appendix 2: Example of Standard Curve Preparation (Data for reference only)
The absorbance results of the 50, 25, 12.5, 6.25, 3.125 and 0 μmol/L gradient samples detected by the absorbance method according to the information in the manual are as follows:
| Concentration （μmol/L): OD532 | 0: 0.042 |
| 3.125: 0.056 | 6.25: 0.069 |
| 12.5: 0.098 | 25: 0.153 |
| 50: 0.267 | Concentration （μmol/L): △OD532 |
| 0: 0 | 3.125: 0.014 |
| 6.25: 0.027 | 12.5: 0.056 |
| 25: 0.111 | 50: 0.225 |
With the sample concentration on the x-axis and the △OD532 results on the y-axis, the fitted equation of the standard curve obtained is Y=0.0045X-0.0005，R2=0.9999.
The absorbance results of the 10, 8, 6, 4, 2, and 0 μmol/L gradient samples detected by the fluorescence intensity method according to the information in the manual are as follows:
| Concentration （μmol/L): OD532 | 0: 357 |
| 2: 2110 | 4: 3841 |
| 6: 5572 | 8: 7304 |
| 10: 9020 | Concentration （μmol/L): △OD532 |
| 0: 0 | 2: 1753 |
| 4: 3484 | 6: 5215 |
| 8: 6947 | 10: 8663 |
With the sample concentration on the x-axis and the △OD532 results on the y-axis, the fitted equation of the standard curve obtained is Y=866.11X-13.095，R2=1.
Appendix 3: Sample Testing Example (Data for reference only)
In accordance with the instruction of absorbance method of operating an example of measuring a cell samples of OD532 is 0.094, and the OD532 of the blank sample is 0.042. The calculated △OD532 for this sample is 0.052. When using single well calculation, based on the △OD532 of the 50 μmol/L standard, which is 0.225, the concentration of MDA concentration in the cell sample is calculated as 0.052÷0.225×50=11.556 μmol/L. When using the standard curve for calculation, substituting the △OD532 of this sample, which is 0.052, into the fitting equation of the standard curve, the concentration of MDA concentration is calculated as (0.052+0.0005)÷0.0045=11.667 μmol/L.
In accordance with the instruction of fluorescence intensity method of operating an example of measuring a cell samples of OD532 is 2590, and the OD532 of the blank sample is 357. The calculated △OD532 for this sample is 2233. When using single well calculation, based on the △OD532 of the 4 μmol/L standard, which is 3484, the concentration of MDA concentration in the cell sample is calculated as 2233÷3484×4=2.564 μmol/L. When using the standard curve for calculation, substituting the △OD532 of this sample, which is 2233, into the fitting equation of the standard curve, the concentration of MDA concentration is calculated as (2233+13.095)÷866.11=2.593 μmol/L.
For Research Use Only!
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