**Plant β-Glucuronidase (β-GUS) ELISA Kit – For Research Use Only**
This kit is designed for the quantitative in vitro determination of β-Glucuronidase (β-GUS) levels in plant tissues, cell cultures, and other biological samples. The assay is based on the double-antibody sandwich ELISA method. A purified anti-β-GUS antibody is coated onto microtiter plate wells to create a solid-phase antibody. The sample containing β-GUS is then added, allowing it to bind to the immobilized antibody. After washing, an HRP-conjugated secondary antibody is introduced, forming an antibody-antigen-enzyme complex. TMB substrate is added, resulting in a blue color change catalyzed by HRP. The reaction is stopped with sulfuric acid, and the absorbance is measured at 450 nm. The concentration of β-GUS in the sample is determined by comparing its optical density (OD) to a standard curve.
**Storage:** 2–8°C. **Expiration Date:** 6 months from date of manufacture.
**Kit Components (96 determinations):**
- 1 x User Manual
- 2 x Closure Plate Membrane
- 1 x Sealed Bag
- 1 x MicroELISA Stripplate
- 1 x Standard (900 pg/ml, 0.5 ml)
- 1 x Standard Diluent (1.5 ml)
- 1 x HRP-Conjugate Reagent (6 ml)
- 1 x Sample Diluent (6 ml)
- 1 x Chromogen Solution A (6 ml)
- 1 x Chromogen Solution B (6 ml)
- 1 x Stop Solution (6 ml)
- 1 x Wash Solution (20 ml × 30 fold)
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant. If precipitate forms, re-centrifuge.
- **Plasma:** Use EDTA or citrate as anticoagulant. Mix for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant. If precipitate forms, re-centrifuge.
- **Urine:** Collect in a sterile container, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant. If precipitate forms, re-centrifuge.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. Dilute cell suspension with PBS (pH 7.2–7.4), freeze-thaw repeatedly, then centrifuge again.
- **Tissue Samples:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant. Store at -20°C if not used immediately.
**Important Notes:**
- Avoid using samples containing NaN3, as it inhibits HRP activity.
- Allow all reagents to reach room temperature before use.
- Wash thoroughly after each step to remove unbound components.
- Prepare a standard curve for each test, and ensure accurate pipetting.
- Keep all reagents away from light.
- Do not mix reagents from different batches.
- Follow the manufacturer’s instructions strictly.
**Assay 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 5 times with diluted wash solution.
5. Add HRP-conjugated reagent.
6. Incubate again for 30 minutes.
7. Wash again.
8. Add TMB substrate and incubate for 15 minutes.
9. Stop the reaction with stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Performance Specifications:**
- Correlation coefficient (R) of linear regression ≥ 0.92.
- Intra-assay CV < 9%, Inter-assay CV < 15%.
- Detection range: 10 pg/ml – 600 pg/ml.
**Note:** This kit is intended for research purposes only. Not for diagnostic use. Always follow proper biosafety protocols when handling samples and reagents.
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