**Mouse Matrix Metalloproteinase 1 (MMP-1) ELISA Kit – Instructions for Use**
**Kit Specifications:**
Available in 48-well or 96-well configurations.
Standard Diluent: 1.5 mL × 1 bottle
Enzyme Standard Reagent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
Storage Conditions: 2–8°C
**Calculation Method:**
Plot the standard curve with standard concentrations on the x-axis and OD values on the y-axis. Determine the sample concentration from the curve based on its OD value, then multiply by the dilution factor. Alternatively, use linear regression to calculate the equation of the standard curve and substitute the sample’s OD value into the equation to determine the actual concentration.
**Kit Components:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 bottle, 2700 ng/L
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Developer A: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Chromogen B: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Wash Solution: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Concentrated Wash Solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well)
**Principle of Operation:**
This kit uses a double-antibody sandwich method to detect MMP-1 levels in samples. The microplate is pre-coated with purified anti-MMP-1 antibodies. After adding the sample, HRP-labeled anti-MMP-1 antibodies bind to the antigen, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, and the color changes from blue to yellow under HRP catalysis. The intensity of the color is proportional to the MMP-1 concentration, which is quantified using a standard curve at 450 nm.
**Purpose:**
The kit is designed to measure the concentration of matrix metalloproteinase-1 (MMP-1) in serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Service Commitment:**
- Delivery time: From payment to delivery
- Free technical support during working hours
- Free sample testing services available upon request
- Storage: 2–8°C, Shelf Life: 6 months
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 minutes, collect supernatant.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix for 10–20 minutes, centrifuge, and collect supernatant.
3. **Urine:** Collect in sterile tubes, centrifuge, and carefully collect supernatant.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes, collect supernatant. For intracellular components, lyse cells via freezing and thawing, then centrifuge.
5. **Tissue Specimens:** Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing.
6. **Storage:** Process samples as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
7. **Note:** Avoid using samples containing NaN₃, as it may inhibit HRP activity.
**Procedure Steps:**
1. **Standard Dilution & Loading:** Prepare serial dilutions of the standard and load into designated wells.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample to each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute concentrated wash solution and wash 5 times.
5. **Enzyme Addition:** Add 50 μL of enzyme reagent to each well (except blank).
6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to terminate the reaction.
9. **Measurement:** Read absorbance at 450 nm within 15 minutes.
**Important Notes:**
- Allow the kit to reach room temperature before use.
- If the enzyme reagent is opened, store it in a sealed bag.
- Wash solution may crystallize; heat gently if needed.
- Use accurate pipettes and avoid cross-contamination.
- Always run standards in duplicate.
- Keep substrates away from light.
- Follow instructions strictly and verify results with a microplate reader.
- Treat all waste materials as biohazardous.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
This guide ensures accurate and reliable detection of MMP-1 in various biological samples. Always follow safety protocols and maintain proper storage conditions for optimal performance.
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