**Human Arginase (Arg) ELISA Kit Protocol**
Before starting the experiment, make sure to prepare all reagents in advance. When diluting samples or reagents, mix thoroughly and avoid any contamination. Always use a standard curve for each test. If the sample concentration is too high, dilute it with the provided sample diluent to ensure it falls within the detection range of the kit.
1. **Sample Addition**:
Add 100 µl of sample diluent to the blank wells, and 100 µl of standard or sample to the respective wells. Ensure no air bubbles are introduced. Carefully add the sample to the bottom of each well, avoiding contact with the walls. Gently mix and cover the plate with a lid. Incubate at 37°C for 120 minutes.
It's important to use a fresh standard solution for every experiment to ensure accuracy.
2. **Biotinylated Antibody Addition**:
After incubation, discard the liquid and gently dry the plate. Add 100 µl of biotinylated antibody working solution per well (prepared by mixing 1 µl of biotinylated antibody with 99 µl of diluent). Mix gently and incubate at 37°C for 60 minutes.
3. **Washing Step**:
Remove the liquid and dry the plate. Wash the plate 3 times with wash buffer, soaking each well for 1–2 minutes. Ensure complete removal of liquid after each wash.
4. **HRP-Conjugated Avidin Addition**:
Add 100 µl of horseradish peroxidase (HRP)-labeled avidin working solution to each well. Incubate at 37°C for 60 minutes.
5. **Final Washing**:
Discard the liquid, dry the plate, and wash it 5 times with wash buffer. Soak each well for 1–2 minutes and ensure thorough drying.
6. **Substrate Addition**:
Add 90 µl of substrate solution to each well. Avoid exposure to light and observe color development. Within 30 minutes, the first 3–4 standard wells should show a clear blue gradient. If the color becomes too intense, stop the reaction immediately.
7. **Stop Solution Addition**:
Add 50 µl of stop solution to each well to terminate the reaction. The color will turn yellow. Add the stop solution as soon as possible after the substrate incubation time to maintain accuracy.
8. **OD Measurement**:
Measure the optical density (OD) at 450 nm using an ELISA reader. Perform the measurement within 15 minutes of adding the stop solution to ensure accurate results.
**Notes for Human Arginase (Arg) ELISA Kit**:
1. For first-time use, centrifuge all reagent tubes briefly to collect the reagents at the bottom of the tube.
2. Reserve one well as a blank zero well for baseline adjustment. This well should only contain the final substrate and stop solution.
3. To prevent evaporation, cover the plate with a lid or film and place it in a closed container with a damp cloth.
4. Store unused plates and reagents at 2–8°C. Do not reuse diluted standards, biotinylated antibodies, or HRP-labeled avidin solutions.
5. It is recommended to run samples in duplicate to improve the reliability of the results.
This detailed protocol ensures accurate and reproducible results when measuring human arginase levels using the ELISA kit. Always follow the manufacturer’s instructions carefully and maintain a clean and controlled experimental environment.
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