**Mouse Tyrosine Hydroxylase (TH) ELISA Kit – User Manual** This kit is intended for research use only and is designed to quantify tyrosine hydroxylase (TH) in mouse serum, plasma, and other biological fluids. The method employed is a double-antibody sandwich ELISA, ensuring high specificity and sensitivity. **Principle of the Assay** The microplate is pre-coated with a specific antibody against mouse TH. After adding the sample, the target antigen binds to the immobilized antibody. A second HRP-conjugated antibody then recognizes the captured antigen, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is added, which turns blue under HRP catalysis and then yellow upon acid termination. The intensity of the color correlates with the concentration of TH in the sample. Absorbance is measured at 450 nm using a microplate reader, and the TH concentration is calculated based on a standard curve. **Kit Components** - 48-well or 96-well configuration - Microplate coated with anti-TH antibody - Standard: 900 pg/mL (0.5 mL × 1 bottle) - Standard diluent: 1.5 mL × 1 bottle - Enzyme-labeled reagent: 3 mL × 1 bottle - Sample diluent: 3 mL × 1 bottle - TMB Substrate A & B: 3 mL × 1 bottle each - Wash buffer (20×): 20 mL × 20 times or 30 times - Sealing film and bag: 2 pieces per kit **Storage Instructions** All components should be stored at 2–8°C. The kit is valid for 6 months from the date of receipt. **Sample Preparation** - **Serum**: Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. - **Plasma**: Use EDTA, citrate, or heparin as anticoagulant. Centrifuge after mixing. - **Urine**: Collect in sterile tubes and centrifuge. - **Cell culture supernatant**: Centrifuge after collection. For intracellular proteins, lyse cells by freeze-thaw cycles. - **Tissue**: Homogenize in PBS, centrifuge, and collect supernatant. **Procedure** 1. Prepare standards by serial dilution (600, 400, 200, 100, 50 pg/mL). 2. Add 40 μL sample diluent and 10 μL sample to each well. 3. Seal the plate and incubate at 37°C for 30 minutes. 4. Wash 5 times with diluted wash buffer. 5. Add 50 μL enzyme-labeled reagent to all wells except blanks. 6. Incubate again at 37°C for 30 minutes. 7. Wash thoroughly. 8. Add 50 μL TMB A and B, incubate at 37°C for 15 minutes. 9. Stop the reaction with 50 μL stop solution. 10. Measure OD at 450 nm within 15 minutes. **Notes** - Allow the kit to equilibrate to room temperature before use. - Avoid cross-contamination by using a new sealing film for each test. - Always prepare a standard curve and run duplicates. - If the sample OD exceeds the highest standard, dilute and retest. - Handle all reagents carefully and dispose of waste as biohazard. - Do not mix components from different batches. **Calculation** Plot the standard curve using concentration vs. OD values. Determine the sample concentration from the curve and multiply by the dilution factor. **Performance** - Intra- and inter-assay coefficients of variation <9% and <11%, respectively. - Linear range: 30–700 pg/mL. This manual provides a comprehensive guide for accurate and reliable detection of mouse TH levels. Always follow the instructions carefully and ensure proper handling of all materials.

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