**Plant β-Glucuronidase (β-GUS) ELISA Kit – For Research Use Only**
This ELISA kit is designed for the quantitative in vitro determination of β-glucuronidase (β-GUS) levels in plant tissues, cell cultures, and other biological samples. It utilizes a double-antibody sandwich immunoassay technique to detect and quantify β-GUS with high specificity and sensitivity. The kit has a shelf life of 6 months when stored at 2–8°C.
**Principle of the Assay**
The assay involves coating microtiter plate wells with purified anti-β-GUS antibodies. After incubation, the sample is added, allowing β-GUS to bind to the immobilized antibodies. A horseradish peroxidase (HRP)-labeled secondary antibody is then introduced, forming an antibody-antigen-enzyme complex. Following thorough washing, TMB substrate is added, which turns blue under HRP catalysis. The reaction is stopped by adding sulfuric acid, causing the color to change to yellow. The absorbance is measured spectrophotometrically at 450 nm, and the β-GUS concentration is determined by comparing the sample OD values to a standard curve.
**Kit Components**
- 96-well microplate
- 1 User Manual
- 2 Closure Plate Membranes
- 1 Sealed Bag
- 1 MicroELISA Strip Plate
- 1 Standard (900 pg/ml, 0.5 ml)
- 1 Standard Diluent (1.5 ml)
- 1 HRP-Conjugate Reagent (6 ml)
- 1 Sample Diluent (6 ml)
- 1 Chromogen Solution A (6 ml)
- 1 Chromogen Solution B (6 ml)
- 1 Stop Solution (6 ml)
- 1 Wash Solution (20 ml × 30-fold, 1 bottle)
**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 and re-centrifuge if precipitate forms.
- **Plasma**: Use EDTA or citrate as anticoagulant. Mix for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant and re-centrifuge if needed.
- **Urine**: Collect in a sterile container, centrifuge at 2000–3000 rpm for 20 minutes. Remove supernatant and re-centrifuge if necessary.
- **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. If intracellular components are being tested, dilute the cell suspension in PBS (pH 7.2–7.4), lyse by freeze-thaw cycles, and centrifuge again.
- **Tissue Samples**: Weigh the tissue, add PBS (pH 7.4), homogenize, and centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant for testing.
**Important Notes**
- Always allow the kit components to equilibrate to room temperature before use.
- Avoid repeated freeze-thaw cycles for samples. Store at -20°C if not used immediately.
- Do not use samples containing NaN3, as it inhibits HRP activity.
- Maintain strict protocol adherence to ensure accurate results.
- All waste materials should be treated as biohazardous.
- Do not mix reagents from different batches.
**Assay Procedure**
1. Prepare standard dilutions and add to designated wells.
2. Add sample diluent and test samples to appropriate wells.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash solution.
5. Add HRP-conjugated reagent to all wells except blank.
6. Incubate again for 30 minutes.
7. Add chromogen solutions A and B, incubate for 15 minutes in the dark.
8. Stop the reaction by adding stop solution.
9. Measure absorbance at 450 nm within 15 minutes.
**Data Analysis**
Plot the standard curve using the OD values against known concentrations. Calculate the unknown sample concentrations using linear regression. Ensure that the correlation coefficient (R²) is ≥ 0.92 for reliable results. Intra-assay and inter-assay variations should be less than 9% and 15%, respectively.
**Storage and Shelf Life**
Store all components at 2–8°C. The kit is valid for 6 months from the date of manufacture.
**Limitations**
This kit is intended for research purposes only and is not suitable for diagnostic or clinical use.
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