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Rat Heat Shock Factor 1 (HSF1) ELISA Kit Instructions for Use
**Rat Heat Shock Factor 1 (HSF1) ELISA Kit – User Manual**
This ELISA kit is designed for the quantitative determination of Rat Heat Shock Factor 1 (HSF1) in biological samples such as serum, plasma, urine, cell culture supernatants, and tissue homogenates. It utilizes a sandwich immunoassay technique with high sensitivity and specificity.
**Kit Specifications:**
- **Configuration:** 48-well or 96-well format
- **Standard Dilution:** 1.5 mL × 1 vial (for 48-well), 3 mL × 1 vial (for 96-well)
- **Enzyme Standard Reagent:** 3 mL × 1 vial (48), 6 mL × 1 vial (96)
- **Storage Conditions:** 2–8°C
- **Shelf Life:** 6 months from date of manufacture
**Principle of Operation:**
The kit employs a double-antibody sandwich ELISA method. The microtiter plate is pre-coated with a specific antibody against HSF1. After adding the sample and standards, HSF1 binds to the immobilized antibody. An enzyme-conjugated secondary antibody is then added, forming a complex. TMB substrate is used for color development, which changes from blue to yellow upon addition of the stop solution. The optical density (OD) at 450 nm is measured, and the concentration of HSF1 in the sample is determined by comparing it to a standard curve.
**Sample Preparation Guidelines:**
- **Serum/Plasma:** Centrifuge at 2000–3000 rpm for 20 minutes after clotting or anticoagulant addition.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge to remove debris; for intracellular components, lyse cells via freezing/thawing.
- **Tissue Homogenate:** Homogenize in PBS, centrifuge, and collect the supernatant.
**Procedure Summary:**
1. **Standard Dilution:** Prepare a serial dilution of the standard from 1800 ng/L down to 150 ng/L.
2. **Sample Loading:** Add 40 µL of sample diluent followed by 10 µL of sample to each well (final dilution 5×).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted washing buffer (1:30 dilution of concentrated solution).
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 µL of TMB A and B, incubate for 15 minutes at 37°C.
8. **Stop Reaction:** Add 50 µL of stop solution to each well.
9. **Measurement:** Read OD values at 450 nm within 15 minutes.
**Important Notes:**
- Allow all reagents to equilibrate to room temperature before use.
- Avoid repeated freeze-thaw cycles of samples.
- Do not mix reagents from different batches.
- Keep the substrate away from light.
- Always run a standard curve in duplicate for accurate results.
- Dispose of all waste as biohazardous material.
**Performance Characteristics:**
- **Sensitivity:** 0.2 IU/L
- **Dynamic Range:** 0.2–6 IU/L
- **Correlation Coefficient (R²):** ≥ 0.95
- **Intra-batch CV:** < 9%
- **Inter-batch CV:** < 11%
**Company Commitment:**
Jianglai Bio provides free technical support during working hours, and offers sample testing services to ensure optimal performance. We guarantee delivery within the agreed time frame and support you throughout your research process.
**Note:** This product is for research use only. Not for diagnostic or therapeutic purposes.