**Rat c-Jun N-terminal Kinase (JNK) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed to quantify the level of c-Jun amino-terminal kinase (JNK) in rat serum, plasma, and other biological fluids. The assay is based on a double-antibody sandwich ELISA method. A microplate pre-coated with a specific anti-JNK antibody is used as the solid phase. After adding the sample, the JNK present in the sample binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured JNK, forming an antibody-antigen-enzyme complex. Following washing steps, a TMB substrate is added, which changes color in the presence of HRP. The reaction is stopped with an acidic solution, and the absorbance is measured at 450 nm. The intensity of the color is directly proportional to the concentration of JNK in the sample.
**Kit Components (96-well format):**
- 1 × 96-well plate
- 2 sealing films
- 1 standard (22.5 ng/ml)
- 1 standard diluent (1.5 ml)
- 1 enzyme-labeled reagent (6 ml)
- 1 sample diluent (6 ml)
- 1 developer A (6 ml)
- 1 developer B (6 ml)
- 1 wash buffer (concentrated, 20×, 20 ml × 30)
- 1 storage bag
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of manufacture.
**Sample Preparation Guidelines:**
- **Serum:** Collect blood at room temperature, allow clotting for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix well, and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes after collection.
- **Cell culture supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freeze-thaw cycles before centrifugation.
- **Tissue samples:** Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant.
**Important Notes:**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN₃, as it inhibits HRP activity.
- Always prepare a standard curve with duplicate wells for accurate quantification.
- Ensure all reagents are equilibrated to room temperature before use.
- Keep the substrate away from light during incubation.
- Discard any unused portions of the sealing film to prevent cross-contamination.
**Procedure Summary:**
1. Prepare serial dilutions of the standard.
2. Add 40 µL of sample diluent and 10 µL of sample to each test well.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add 50 µL of HRP-conjugated antibody to each well.
6. Incubate again for 30 minutes.
7. Wash thoroughly.
8. Add 50 µL of TMB A and B, incubate for 15 minutes.
9. Stop the reaction with 50 µL of stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Data Analysis:**
Plot the standard curve using concentrations vs. OD values. Calculate the sample concentration using linear regression and multiply by the dilution factor.
**Performance:**
- Intra- and inter-assay coefficients of variation <9% and <15%, respectively.
- Linear range: 0.6 ng/mL to 20 ng/mL.
- Correlation coefficient (R²) ≥ 0.92.
**Safety & Disposal:**
Treat all waste materials as biohazardous. Follow local regulations for disposal.
This kit provides a reliable and sensitive method for measuring JNK levels in various biological samples. Always follow the instructions carefully to ensure accurate results.
Sofa Sets,sofa set for living room,recliner sofa set,leather sofa set
Guangzhou LoPhiDa Co.Ltd , https://www.gzwidinlsa.com