**Rat TAU Protein (TAU) ELISA Kit Instruction Manual**
This reagent is intended for research use only and is designed to quantify TAU protein in rat serum, cell culture supernatants, and other liquid samples. The assay is based on the double-antibody sandwich ELISA method. A microplate coated with a specific monoclonal antibody against rat TAU is used as the solid phase. After incubation with the sample, the TAU protein binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an antibody-antigen-enzyme complex. Following washing, the substrate TMB is introduced, which changes color in the presence of HRP. The reaction is stopped with an acidic solution, causing the color to shift from blue to yellow. The intensity of the color is directly proportional to the TAU concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the TAU levels are determined by comparing the sample OD values to a standard curve.
**Kit Components:**
- 48-well or 96-well configuration
- Sealing film (2 pieces per kit)
- Enzyme-labeled plate (1×48 or 1×96)
- Standard (270 ng/L, 0.5 ml × 1 bottle)
- Standard diluent (1.5 ml × 1 bottle)
- Enzyme reagent (3 ml × 1 bottle)
- Sample diluent (3 ml × 1 bottle)
- TMB Developer A and B (3 ml × 1 bottle each)
- Wash buffer (20× concentrate, 20 ml × 20 times or 30 times)
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months when properly stored.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant and re-centrifuge if precipitates form.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix thoroughly and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
- **Tissue Samples:** Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. Store at 2–8°C after thawing.
- **General:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freeze-thaw cycles. Note: Sodium azide (NaN3) must not be present in samples, as it inhibits HRP activity.
**Procedure Summary:**
1. Prepare standards by serial dilution.
2. Add samples and blanks to the plate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate five times with diluted wash buffer.
5. Add HRP-conjugated antibody and incubate again.
6. Add TMB substrate and develop color for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure OD at 450 nm.
9. Calculate concentrations using a standard curve.
**Important Notes:**
- Allow the kit to reach room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Ensure accurate pipetting and maintain consistent timing.
- Always run a standard curve and consider diluting high-concentration samples.
- Handle all waste as biohazardous material.
- Do not mix reagents from different batches.
**Performance Specifications:**
- Correlation coefficient (R²) ≥ 0.92
- Intra-assay CV < 9%, Inter-assay CV < 15%
- Detection range: 5 ng/L – 200 ng/L
This manual provides detailed instructions for accurate and reliable TAU quantification in rat biological samples. Always follow the protocol carefully and refer to the manufacturer’s guidelines for best results.
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