**Rat TAU Protein (TAU) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual**
This reagent is intended for research use only. The kit is designed to quantify Rat TAU protein (TAU) in various biological samples, including rat serum, cell culture supernatants, and other liquid specimens.
**Principle of the Assay**
The assay is based on the double-antibody sandwich ELISA method. A microplate is pre-coated with a specific antibody against Rat TAU. After incubation with the sample, the TAU protein binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, the substrate TMB is introduced, which changes color under the catalytic action of HRP. The reaction is stopped with an acidic solution, and the final color intensity is directly proportional to the TAU concentration in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the TAU concentration is determined by comparing the sample OD to a standard curve.
**Kit Components**
- Microtiter Plate: 1 × 48 or 1 × 96 wells
- Standard: 0.5 mL × 1 bottle (270 ng/L)
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Conjugate: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Stop Solution: 3 mL × 1 bottle
- Wash Buffer (20×): 20 mL × 20 times or 20 mL × 30 times
- Sealing Film: 2 pieces
- Sealed Bag: 1 piece
**Storage Instructions**
Store all components at 2–8°C. The kit is stable for 6 months from the date of manufacture.
**Sample Preparation**
1. **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 precipitate forms.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge similarly.
3. **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
4. **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freeze-thaw cycles before centrifugation.
5. **Tissue Samples**: Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant.
6. Avoid repeated freezing and thawing; store samples at -20°C if not tested immediately. Do not use samples containing NaN3.
**Procedure**
1. Prepare standard dilutions in triplicate.
2. Add 40 μL of sample diluent followed by 10 μL of sample to each well (final dilution 5×).
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add 50 μL of enzyme conjugate to each well except blank.
6. Incubate again at 37°C for 30 minutes.
7. Wash thoroughly.
8. Add 50 μL of TMB A and B, incubate for 15 minutes at 37°C in the dark.
9. Add 50 μL of stop solution to each well.
10. Measure OD450 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 test.
- Ensure accurate pipetting and avoid light exposure during TMB development.
- Always run standards in duplicate and prepare a standard curve for accurate quantification.
- Discard all waste materials as biohazardous.
**Calculation**
Plot the standard concentrations against their OD values to create a standard curve. Calculate the sample concentration using linear regression, and multiply by the dilution factor.
**Performance**
- Intra-assay CV <9%, Inter-assay CV <15%.
- Linear range: 5 ng/L – 200 ng/L.
- Correlation coefficient (R²) ≥ 0.92.
This manual provides detailed guidance for accurate and reliable measurement of Rat TAU levels in biological samples. Always follow the instructions carefully for best results.
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