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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**
**Kit Specifications:**
This kit is available in a 48-well or 96-well configuration. The standard dilution is 1.5 ml per bottle, with the enzyme standard reagent provided as 3 ml (for 48-well) or 6 ml (for 96-well). The kit is for research use only. The standard curve is plotted by plotting the concentration of the standard on the x-axis and the OD value on the y-axis. The sample concentration can be determined by either reading from the standard curve or calculating the linear regression equation and using it to determine the sample concentration.
**Kit Components:**
- Sealing membranes: 2 pieces (48-well), 2 pieces (96-well)
- Instruction manual: 1 piece
- Standard: 0.5 ml × 1 bottle at 2700 ng/L
- Enzyme-labeled coating plate: 1 × 48, 1 × 96
- Sample diluent: 3 ml × 1 bottle (48), 6 ml × 1 bottle (96)
- Developer A: 3 ml × 1 bottle, 6 ml × 1 bottle
- Developer B: 3 ml × 1 bottle, 6 ml × 1 bottle
- Wash solution: 3 ml × 1 bottle, 6 ml × 1 bottle
- Concentrated wash solution: 20 ml × 20 times (for 48-well), 20 ml × 30 times (for 96-well)
**Storage Conditions:**
- Store the kit at 2–8°C
- Shelf life: 6 months from the date of manufacture
**Principle of the Assay:**
This ELISA kit uses a sandwich method to detect Rat Heat Shock Factor 1 (HSF1). The microplate is pre-coated with a specific antibody against HSF1. After adding the sample, the target antigen binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. TMB substrate is used to develop color, which is stopped by adding an acidic solution. The intensity of the color is directly proportional to the concentration of HSF1 in the sample, measured at 450 nm.
**Objective:**
This kit is designed to quantify HSF1 levels in rat serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix well, centrifuge, and collect the supernatant.
- **Urine:** Collect in sterile tubes and centrifuge to remove debris.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells with PBS and centrifuge again.
- **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
- **Storage:** Samples should be processed immediately. If not tested right away, store at -20°C, avoiding repeated freeze-thaw cycles. Note: Avoid samples containing NaN3, as it may inhibit HRP activity.
**Assay Procedure:**
1. **Standard Dilution and Loading:** Prepare serial dilutions of the standard and add to designated wells.
2. **Sample Addition:** Add 40 μL of sample diluent and 10 μL of sample to each test well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash the plate 5 times with diluted washing solution.
5. **Enzyme Conjugate Addition:** Add 50 μL of enzyme-labeled reagent to each well (except blank).
6. **Second Incubation:** Repeat the incubation step.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Reading:** Measure absorbance at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate to room temperature before use.
- Ensure all reagents are properly mixed and handled.
- Use separate pipettes for each step to avoid contamination.
- Always prepare a standard curve and perform duplicate measurements.
- Avoid exposure of substrates to light.
- Follow the manufacturer’s instructions strictly for accurate results.
- All waste materials should be treated as biohazardous.
**Performance Characteristics:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-batch and inter-batch variation: <9% and <11%, respectively
For technical support, sample testing, and detailed guidance, please contact Jianglai Bio-China. We are committed to providing reliable products and services to ensure successful experimental outcomes.