Guangrui Bio - China's Leading Supplier of High-Quality ELISA Kits for Rat Cholesterol (CH): Technical Manual Procedure
1. **Preparation and Dilution of Standard Solutions**: Begin by setting up 10 standard wells on the ELISA plate. Add 100 μl of the standard solution to the first and second wells, then add 50 μl of the standard diluent to each. Mix thoroughly. Next, transfer 100 μl from the first and second wells into the third and fourth wells, respectively, and add 50 μl of diluent to each. Mix again. Discard 50 μl from the third and fourth wells, then add 50 μl of the mixture to the fifth and sixth wells. Add 50 μl of diluent to the fifth and sixth wells, mix, and transfer 50 μl from each to the seventh and eighth wells. Finally, add 50 μl of diluent to the seventh and eighth wells, mix, and transfer 50 μl to the ninth and tenth wells. After final mixing, discard 50 μl from the ninth and tenth wells. Each well now contains 50 μl with concentrations of 30, 20, 10, 5, and 2.5 μmol/L, respectively.
2. **Sample Addition**: Set up blank control wells (no sample or enzyme reagent added), and prepare the test wells. Add 40 μl of sample diluent to each test well, followed by 10 μl of the sample. This results in a 5-fold dilution. Carefully pipette the sample to the bottom of the well, avoiding contact with the walls, and gently mix.
3. **Incubation**: Seal the plate with an adhesive film and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation**: Dilute the concentrated washing buffer (30 times for 20T, 20 times for 48T) with distilled water as specified.
5. **Washing**: Remove the seal, discard the liquid, and blot dry. Fill each well with washing solution, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat the plate dry.
6. **Enzyme Conjugate Addition**: Add 50 μl of enzyme-labeled reagent to each well except the blank controls.
7. **Second Incubation**: Repeat the same incubation procedure as step 3: seal the plate and incubate at 37°C for 30 minutes.
8. **Second Washing**: Follow the same washing steps as in step 5.
9. **Color Development**: Add 50 μl of color reagent A, followed by 50 μl of color reagent B. Gently mix the solution and avoid exposure to light. 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. **Measurement**: Measure the absorbance (OD value) at 450 nm using a microplate reader. Ensure measurements are taken within 15 minutes after adding the stop solution to ensure accuracy.
This detailed procedure ensures precise quantification of rat cholesterol levels using the ELISA kit. Always follow the manufacturer’s instructions and maintain proper lab practices for reliable results.
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