**Rat c-Jun N-terminal Kinase (JNK) ELISA Kit – User Manual**
*For Research Use Only*
This ELISA kit is designed for the quantitative determination of c-Jun N-terminal kinase (JNK) in rat serum, plasma, and other biological fluids. The method employed is a double-antibody sandwich ELISA, which ensures high specificity and sensitivity for JNK detection.
**Principle of the Assay**
The assay utilizes a microtiter plate pre-coated with a specific antibody against rat JNK. After incubation with the sample, JNK binds to the immobilized antibody. A secondary HRP-conjugated antibody then recognizes the captured JNK, forming an immune complex. The addition of TMB substrate leads to a color change, which is proportional to the JNK concentration in the sample. The reaction is stopped by adding an acidic solution, turning the color from blue to yellow. The optical density (OD) at 450 nm is measured and correlated with JNK levels via a standard curve.
**Kit Components**
- Microtiter Plate: 48 or 96 wells
- Standard: 22.5 ng/mL, 0.5 mL × 1 bottle
- 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 Films: 2 pieces per kit
- Storage: 2–8°C
**Sample Preparation Guidelines**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant; centrifuge after mixing.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant**: Centrifuge after collection; for intracellular components, lyse cells via freeze-thaw cycles.
- **Tissue Homogenate**: Homogenize in PBS, centrifuge, and collect supernatant.
**Precautions**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it may inhibit HRP activity.
- All reagents should be brought to room temperature before use.
- Discard any unsealed strips after opening and store in a sealed bag.
- Always include blank controls and prepare standard curves in duplicate.
**Procedure Summary**
1. Prepare serial dilutions of the standard.
2. Add 40 µL sample diluent and 10 µL sample to each well.
3. Seal and incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add 50 µL enzyme conjugate to each well.
6. Incubate again for 30 minutes.
7. Add 50 µL TMB A and B, incubate for 15 minutes.
8. Stop the reaction with stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Data Analysis**
Plot the standard curve using concentration vs. OD values. Calculate the sample concentration using linear regression and multiply by the dilution factor if applicable. Ensure accuracy by performing replicates and checking linearity.
**Storage and Shelf Life**
Store the kit at 2–8°C. The shelf life is 6 months from the date of manufacture.
**Note**
Always follow the manufacturer’s instructions carefully. This manual is intended for research purposes only. For any discrepancies, the English version shall prevail.
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