Total Iron Ion Assay Kit servicebio G4301-96T
$50.00
In stock
SKU
G4301-96T
Product Information
| Product Name: Total Iron Ion Assay Kit | Cat. No.: G4301-48T |
| Spec.: 48 T | G4301-96T |
| 96 T |
Product Description/Introduction
Iron is one of the important metallic elements in living organisms, participating in various physiological activities such as hematopoiesis, oxygen transport, enhancing immunity, and the synthesis and reactivity of certain enzymes. With the proposal of the concept of ferroptosis in recent years, the change in iron ion content, as one of the characteristics of ferroptosis, also indicates that the iron ion metabolic process has an important relationship with the health and disease of organisms.
This product is a Total Iron Assay Kit. The detection principle is that under special conditions, iron ions in the sample are fully released and rapidly reduced to ferrous ions by the reducing agent in the reagent. Subsequently, ferrous ions combine with the detection probe to form colored substances. Within a certain concentration range, the depth of the color of the generated substances is directly proportional to the concentration of iron ions.
Storage and Shipping Conditions
Ship with wet ice; Store at 2-8℃ away from light for 6 months.
Product Content
| Component Number: G4301-1 | Component: Iron ion standard (1 mmol/L) |
| G4301-48T: 0.5 mL | G4301-96T: 0.5 mL |
| Component Number: G4301-2 | Component: Total Iron Detection Buffer |
| G4301-48T: 20 mL | G4301-96T: 40 mL |
| Component Number: G4301-3 | Component: Total Iron Detection Probe |
| G4301-48T: 5 mL | G4301-96T: 10 mL |
| Component Number: G4301-4 | Component: Extract solution |
| G4301-48T: 25 mL | G4301-96T: 50 mL |
Note: The total iron detection buffer and extract solution should be balanced at room temperature for 30 minutes before use.
Assay Protocol / Procedures
1. Sample Preparation:
2. Plasma and serum samples: Thoroughly mix the sample and the extract solution in a ratio of 1:4 for subsequent total iron ion detection.
3. Tissue samples: Please use the extract solution for homogenization and lysis. The ratio of tissue to extract should be 1:9 (for example, 30 mg: 270 μL). After homogenization and lysis at room temperature, centrifuge at 4℃ and 10,000 g for 10-15 minutes, and take the supernatant for the subsequent detection of total iron ions.
4. Cell or fermentation broth samples: After cell collection, resuspend the cells using the extract solution, approximately adding 300 μL of the extract solution for every 1×106 cells. After homogenization and lysis at room temperature, centrifuge at 4°C and 10,000 g for 10-15 minutes, and take the supernatant for subsequent total iron ion detection.
5. Iron ion reference material: Use ultrapure water to gradient dilute the iron ion standard (1 mmol/L). For instance, take 50 μL of it and add it to 950 μL of ultrapure water to dilute it to 50 μmol/ L. Then, further gradient dilute it to create a standard curve, which is 50/25/12.5/6.25/3.125 μmol/L. After subsequent testing with the sample to be tested, Used for data analysis by the standard curve method; Or it can be appropriately diluted to a 25 μmol/L standard substance, and after subsequent testing with the sample to be tested, it can be used for data analysis by the standard substance conversion method;
5.1. Sample testing (operated in a centrifuge tube):
Mix well and incubate at 37°C for 15 minutes
Incubate at 37°C for 40 minutes, centrifuge at 10,000 g for 5 minutes, take 200 μL of the supernatant in an microplate, and measure the absorbance at 593 nm with an microplate reader
| standard tube | blank tube |
| sample tube | Iron ion standard |
| 100 μL | Total Iron Detection Buffer |
| 100 μL | Samples to be tested |
| 100 μL | Total Iron Detection Buffer |
| 100 μL | 100 μL |
| 100 μL | Total Iron Detection Probe |
| 100 μL | 100 μL |
| 100 μL |
6. Data analysis:
3.1 Standard hole calculation method：
Total iron ion content in serum/plasma (μmol/L)=(Asample-Ablank)÷(Astandard-Ablank)×Cstandard×f
Total iron ion content in tissues/cells (μmol/g prot)=(Asample-Ablank)÷(Astandard-Ablank) ×Cstandard÷Cpr
Note: C standard, 25 μmol/L. Cpr: Tissue (cell) homogenate protein concentration, g prot/L. f: The sample dilution ratio is 5.
3.2 Standard curve method:Taking the concentration gradient of the standard substance as the abscissa x and △OD593 as the ordinate y, a standard curve y=ax+b was fabricated.
Total iron ion content in serum/plasma (μmol/L)=(Asample-Ablank-b)÷a×f
Total iron ion content in tissues/cells (μmol/g prot)=(Asample-Ablank-b)÷a÷Cpr
Note: Cpr: Tissue (cell) homogenate protein concentration, g prot/L. f: The sample dilution ratio is 5.
Note
1. Gently shake all reagents before use to ensure that all components are evenly distributed.
2. When analyzing data by the standard conversion method, the concentrations of the sample to be tested and the standard must be within the linear detection range of the kit. When analyzing data by the standard curve method, ensure that the R2 of the standard curve is greater than 0.99.
3. Avoid handling or transferring samples with iron items.
4. For your health and safety, please wear lab coat and gloves during operation.
Appendix 1: Technical parameters
| Detection index: Sample type | Total iron ion concentration: Serum, tissue, cells, culture supernatant and fermentation broth |
| Recovery Rate: Intra-batch CV | 100±10%: <5% |
| Detection index: Detection Sensitivity and Range | Total iron ion concentration: 0.762 μmol/L；0.762-100 μmol/L |
| Recovery Rate: Inter-batch CV | 100±10%: <5% |
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): OD593 | 0: 0.042 |
| 3.125: 0.056 | 6.25: 0.069 |
| 12.5: 0.098 | 25: 0.153 |
| 50: 0.267 | Concentration （μmol/L): △OD593 |
| 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 △OD593 results on the y-axis, the fitted equation of the standard curve obtained is Y=0.0039X+0.001，R2=0.9999.
Appendix 3: Sample Testing Example (Data for reference only)
In accordance with the instruction of absorbance method of operating an example of measuring a serum samples of OD593 is 0.074, and the OD593 of the blank sample is 0.047. The calculated △OD593 for this sample is 0.027. When using single well calculation, based on the △OD593 of the 25 μmol/L standard, which is 0.1, the concentration of Total iron ion concentration in the serum sample is calculated as 0.027÷0.1×25×5=33.750 μmol/L. When using the standard curve for calculation, substituting the △OD593 of this sample, which is 0.027, into the fitting equation of the standard curve, the concentration of Total iron ion concentration is calculated as (0.027-0.001)÷0.0039×5=33.335 μmol/L.
For Research Use Only!
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