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Mouse acidic fibroblast growth factor 1 (aFGF-1) elisa kit instruction manual
**Mouse Acidic Fibroblast Growth Factor 1 (aFGF-1) ELISA Kit – Instructions for Use**
**Kit Specifications:**
This ELISA kit is available in a 48-well or 96-well configuration. The standard dilution is 1.5 mL × 1 vial. The enzyme standard reagent is 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well). This reagent is intended for research use only.
**Standard Curve Preparation:**
To determine the concentration of aFGF-1, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Draw a standard curve on graph paper, and determine the sample concentration based on its OD value. Alternatively, calculate the linear regression equation from the standard curve data and substitute the sample OD value into the equation to find the actual concentration, then multiply by the dilution factor.
**Kit Composition:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial, 2700 ng/L
- Enzyme Standard: 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- Sample Diluent: 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- Developer A: 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- Chromogen B: 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- Wash Solution: 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- Concentrated Wash Solution: 20 mL × 20 times (48), 20 mL × 30 times (96)
**Storage Conditions:**
All components should be stored at 2–8°C. The shelf life of the kit is 6 months from the date of receipt.
**Principle of Operation:**
The kit utilizes a sandwich ELISA method to detect mouse acidic fibroblast growth factor 1 (aFGF-1). A purified anti-aFGF-1 antibody is coated onto microtiter plates. After incubation with the sample, the target protein binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. TMB substrate is used for color development, and the reaction is stopped with a stop solution. The absorbance at 450 nm is measured, and the concentration is calculated using a standard curve.
**Objective:**
This kit is designed for the quantitative determination of aFGF-1 in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids from mice.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing.
- **Urine:** Collect in a sterile tube and centrifuge.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via repeated freeze-thaw cycles.
- **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing.
- **General Note:** Avoid repeated freezing and thawing. Do not use samples containing NaN₃, as it inhibits HRP activity.
**Procedure Summary:**
1. Prepare standards by serial dilution.
2. Add samples and standards to the plate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add enzyme-labeled reagent and incubate again.
6. Add TMB substrate and develop color for 15 minutes.
7. Stop the reaction and measure OD at 450 nm within 15 minutes.
**Important Notes:**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Handle all reagents carefully, and discard waste as biohazardous material.
- Ensure accurate pipetting and maintain consistent timing during loading.
- Always perform a standard curve and run duplicates for accuracy.
**Performance Characteristics:**
- Linearity correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Technical Support:**
Free technical support is available during working hours. We also offer free sample testing services to help ensure optimal experimental results.
**Note:** This kit is for research purposes only and not for diagnostic use.