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Rat Bcl-2 Related X Protein (BAX) ELISA Kit – Step-by-Step Procedure
1. **Standard Dilution**: This kit includes one original standard. Prepare a series of dilutions in a small tube according to the provided standard curve chart.
2. **Sample and Standard Loading**: Set up blank wells (no sample or enzyme reagent), standard wells, and sample wells. Add 50 μl of the standard to each standard well, 40 μl of sample diluent to the test wells, and 10 μl of the sample for a final 5-fold dilution. Carefully add the sample to the bottom of the well without touching the walls. Gently mix by shaking.
3. **Incubation**: Seal the microplate with an adhesive film and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation**: Dilute the 20× washing solution with distilled water in a 1:20 ratio before use.
5. **Washing**: Remove the sealing film, discard the liquid, and pat dry. Fill each well with the washing solution, let it sit for 30 seconds, then discard. Repeat this process 5 times and gently pat dry.
6. **Enzyme Conjugate Addition**: Add 50 μl of enzyme-labeled reagent to each well except the blank ones.
7. **Second Incubation**: Seal the plate again and incubate at 37°C for 30 minutes, following the same procedure as step 3.
8. **Second Washing**: Repeat the washing steps described in point 5.
9. **Color Development**: Add 50 μl of the substrate solution to each well, then add another 50 μl to ensure even distribution. Gently shake the plate and incubate at 37°C for 15 minutes.
10. **Stop Reaction**: Add 50 μl of stop solution to each well to halt the reaction. The color will change from blue to yellow.
11. **Absorbance Measurement**: Measure the absorbance (OD value) at 450 nm using a microplate reader. Ensure the measurement is conducted within 15 minutes after adding the stop solution.
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- The shelf life of the Rat BAX ELISA Kit is 6 months from the date of receipt.
This kit is designed for accurate and reliable detection of Bax protein levels in rat samples. Always follow the manufacturer's instructions carefully to ensure optimal performance and reproducibility.
X3S048000DC1H-X
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