**Human Ubiquitin Protein (Ub) ELISA Kit – Instructions for Use**
This ELISA kit is designed for the quantitative determination of human ubiquitin (Ub) in various biological samples, including serum, plasma, urine, cell culture supernatants, and tissue homogenates. The kit utilizes a sandwich ELISA method, which ensures high specificity and sensitivity for detecting Ub levels.
**Kit Specifications:**
- **Configuration:** 48-well or 96-well plates
- **Standard Dilution:** 1.5 mL × 1 vial (Enzyme Standard Reagent: 3 mL × 1 vial for 48-well; 6 mL × 1 vial for 96-well)
- **Storage Conditions:** 2–8°C for all reagents, except chromogen B which should be stored at -20°C
- **Shelf Life:** 6 months from the date of receipt
**Kit Components:**
- Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well)
- Standard: 0.5 mL × 1 vial (2700 ng/L)
- Enzyme Standard: 1×48 or 1×96
- Sample Diluent: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial, Developer B: 3 mL × 1 vial
- Wash Solution: 20 mL × 20 times or 20 mL × 30 times
- Concentrated Washing Solution: 20 mL × 20 times or 20 mL × 30 times
**Principle of Operation:**
The kit employs a double-antibody sandwich ELISA technique. A microtiter plate pre-coated with anti-human Ub antibody is used as the solid phase. After incubation with the sample, the bound Ub is detected using HRP-conjugated anti-Ub antibody. A TMB substrate is added, producing a blue color that turns yellow upon acid termination. The intensity of the color is directly proportional to the concentration of Ub in the sample. Absorbance is measured at 450 nm, and the concentration is calculated based on a standard curve.
**Sample Preparation:**
- **Serum/Plasma:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by freezing and thawing before centrifugation.
- **Tissue Homogenate:** Homogenize in PBS, centrifuge, and collect the supernatant.
**Procedure Summary:**
1. Prepare serial dilutions of the standard.
2. Add 40 μL of sample diluent and 10 μL of sample to each well (final dilution: 5×).
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted washing solution.
5. Add 50 μL of enzyme-labeled reagent.
6. Incubate again at 37°C for 30 minutes.
7. Wash again.
8. Add 50 μL of TMB A and B, incubate for 15 minutes.
9. Stop the reaction with 50 μL stop solution.
10. Read OD values at 450 nm within 15 minutes.
**Notes & Recommendations:**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- Avoid repeated freeze-thaw cycles.
- Always prepare a standard curve and run duplicates.
- If the sample OD exceeds the highest standard, dilute the sample before testing.
- Do not mix reagents from different batches.
- Handle all waste as biohazardous material.
**Performance:**
- Linear regression correlation coefficient (R) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
We offer free technical support during working hours and sample testing services to ensure optimal results.
**Delivery Time:** From payment to delivery.
This kit is intended for research use only and not for diagnostic purposes.
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