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**Rat Bcl-2 Related X Protein (BAX) ELISA Kit – Step-by-Step Procedure**
This ELISA kit is designed for the quantitative detection of Bax protein in rat samples. The procedure involves several precise steps to ensure accurate and reliable results.
1. **Standard Preparation**:
The kit includes a single original standard that can be diluted according to the provided chart. Proper dilution ensures accurate calibration and consistent performance across all test runs.
2. **Sample and Standard Loading**:
Set up blank, standard, and sample wells on the microplate. Add 50 μl of the standard solution to each standard well. For the sample wells, add 40 μl of sample diluent followed by 10 μl of the sample, resulting in a 5-fold final dilution. Ensure that the sample is added carefully to the bottom of the well without touching the sides to avoid contamination or uneven mixing.
3. **Incubation**:
Seal the plate with a sealing film and incubate at 37°C for 30 minutes. This allows the antibodies to bind effectively to the target proteins.
4. **Washing Solution Preparation**:
Dilute the 20× washing buffer with distilled water to a 1× working concentration before use.
5. **Washing Steps**:
After incubation, remove the sealing film and discard the liquid. Rinse each well with the washing solution, let it stand for 30 seconds, then discard. Repeat this process five times to remove unbound reagents thoroughly. Gently pat the plate dry with a paper towel.
6. **Enzyme Conjugate Addition**:
Add 50 μl of enzyme-labeled reagent to each well except the blank control. This step helps in detecting the bound complexes through a colorimetric reaction.
7. **Second Incubation**:
Re-seal the plate and incubate again at 37°C for 30 minutes to allow the enzyme conjugate to bind to the immune complexes.
8. **Second Washing**:
Repeat the washing steps as described in step 5 to remove any excess enzyme conjugate.
9. **Color Development**:
Add 50 μl of substrate solution to each well. Mix gently and incubate at 37°C for 15 minutes. A blue color will develop in proportion to the amount of Bax present.
10. **Stop Reaction**:
Add 50 μl of stop solution to each well to terminate the reaction. The color will change from blue to yellow, indicating the end of the enzymatic reaction.
11. **Absorbance Measurement**:
Measure the absorbance at 450 nm using a microplate reader. Make sure to measure within 15 minutes after adding the stop solution to ensure accuracy.
**Storage and Shelf Life**:
- Store the kit at 2–8°C.
- The shelf life of the kit is 6 months from the date of receipt.
- Always check the expiration date before use and follow the storage instructions carefully to maintain the integrity of the reagents.
This protocol is ideal for researchers conducting studies on apoptosis, cell death mechanisms, or related pathways in rat models. Proper execution of each step ensures high sensitivity and specificity in Bax detection.
X3S048000DC1H-X
Test - uppercase JPG - memory 146K