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Rat hydroxyproline (Hyp) elisa technical specification
**Rat Hydroxyproline (Hyp) ELISA Technical Note**
This reagent is for research use only. The kit is designed to quantify hydroxyproline (Hyp) levels in rat serum, plasma, and other biological fluids.
**Principle of the Assay**
The method employed is a double-antibody sandwich ELISA. A microtiter plate is pre-coated with purified anti-rat Hyp antibodies. After adding the sample and HRP-conjugated Hyp antibody, a complex is formed between the captured Hyp, the primary antibody, and the enzyme-labeled secondary antibody. Following washing steps, TMB substrate is added. Under HRP catalysis, TMB turns blue and then yellow upon acid addition. The color intensity correlates directly with the Hyp concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the concentration is determined from a standard curve.
**Kit Components**
- 48-well or 96-well configuration
- Coated plates: 1 × 48 or 1 × 96
- Standard: 0.5 mL × 1 bottle (1350 μg/L), stored at 2–8°C
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Reagent: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Developer A & B: 3 mL × 1 bottle each
- Wash Buffer (20×): 20 mL × 1 bottle
- Sealing Films: 2 pieces per kit
- Storage: 2–8°C
**Sample Preparation**
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant, mix, centrifuge, and collect supernatant.
3. **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant**: Centrifuge and collect supernatant. For intracellular components, lyse cells by freezing/thawing, centrifuge, and collect supernatant.
5. **Tissue Homogenate**: Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
6. **Storage**: Process samples immediately after collection. Store at -20°C if not tested right away. Avoid repeated freeze-thaw cycles. Do not use samples containing NaN3.
**Procedure**
1. **Standard Dilution**: Prepare a 5-fold serial dilution of standards (900, 600, 300, 150, 75 μg/L).
2. **Sample Addition**: Add 40 μL sample diluent and 10 μL sample (final 5× dilution) to each well.
3. **Incubation**: Seal and incubate at 37°C for 30 minutes.
4. **Washing**: Wash 5 times with diluted wash buffer.
5. **Enzyme Addition**: Add 50 μL enzyme reagent to each well (except blank).
6. **Incubation**: Repeat incubation.
7. **Color Development**: Add 50 μL TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction**: Add 50 μL stop solution.
9. **Measurement**: Read OD at 450 nm within 15 minutes.
**Notes**
- Equilibrate kit at room temperature before use.
- If enzyme reagent is opened, store in a sealed bag.
- Wash buffer may crystallize; heat gently if needed.
- Use separate pipettes for each step.
- Always include a standard curve and perform duplicates.
- Avoid cross-contamination; use a new sealing film per test.
- Keep substrates away from light.
- Follow instructions precisely.
- Treat all waste as biohazardous.
- Do not mix reagents from different batches.
**Calculation**
Plot standard concentrations vs. OD values to generate a standard curve. Calculate sample concentration using linear regression and multiply by dilution factor.
**Performance**
- Intra-assay CV <9%, Inter-assay CV <11%
- Linear range: 50–1000 μg/L
- Correlation coefficient (R²) ≥ 0.990
**Storage and Shelf Life**
- Store at 2–8°C.
- Shelf life: 6 months.
This note provides detailed guidance for accurate and reliable detection of hydroxyproline in rat samples.