**Rabbit Soluble Vascular Endothelial Cell Protein C Receptor (sEPCR) ELISA Kit – User Manual**
**Kit Specifications:**
- 48-well or 96-well configuration
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Standard Reagent: 3 mL × 1 bottle (for 48-well) / 6 mL × 1 bottle (for 96-well)
- Storage Conditions: 2–8°C
This reagent is intended for **research purposes only**. To determine the concentration of sEPCR in your samples, plot a standard curve by using the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Alternatively, calculate the linear regression equation from the standard curve and use it to determine the sample concentration based on its OD value. Multiply the result by the dilution factor to obtain the actual sample concentration.
**Kit Components:**
- Sealing Plate: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 2700 ng/L, 0.5 mL × 1 bottle
- Enzyme-Labeled Coating Plate: 1 × 48 / 1 × 96
- Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- TMB Substrate A: 3 mL × 1 bottle / 6 mL × 1 bottle
- TMB Substrate B: 3 mL × 1 bottle / 6 mL × 1 bottle
- Wash Buffer: 20 mL × 20 times / 20 mL × 30 times (concentrated)
**Principle of the Assay:**
The kit uses a **double-antibody sandwich ELISA** method to quantify soluble vascular endothelial cell protein C receptor (sEPCR) in biological samples. The microtiter plate is pre-coated with a specific antibody against sEPCR. After incubation with the sample, the antigen binds to the immobilized antibody. A second HRP-conjugated antibody is then added, forming an antibody-antigen-enzyme complex. Following washing, TMB substrate is added, which turns blue under HRP activity and becomes yellow when acid is added. The intensity of the color is directly proportional to the sEPCR concentration in the sample. The absorbance is measured at 450 nm, and the concentration is calculated using a standard curve.
**Objective:**
This ELISA kit is designed to measure the level of sEPCR in rabbit serum, plasma, urine, cell culture supernatants, and other body fluids.
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**Storage & Shelf Life:**
- Store at 2–8°C
- Validity: 6 months
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge and collect supernatant.
3. **Urine:** Collect in sterile tubes, centrifuge, and collect supernatant.
4. **Cell Culture Supernatant:** Centrifuge and collect supernatant. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again.
5. **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant.
6. **General Tips:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN₃, as it may inhibit HRP activity.
**Assay Procedure:**
1. **Standard Dilution:** Prepare a serial dilution of the standard solution in 5-fold steps.
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:** Rinse the plate 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well (except blank wells).
6. **Second Incubation:** Incubate 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 each well.
9. **Reading:** Measure OD at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate at room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure accurate pipetting and maintain consistent timing.
- Always prepare a standard curve and run duplicates.
- Keep all reagents away from light.
- Follow the manual strictly for reliable results.
- Dispose of all waste as biohazardous material.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Performance Characteristics:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
This kit offers a reliable and sensitive method for detecting sEPCR in various biological matrices. Proper handling and adherence to the protocol will ensure accurate and reproducible results.
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