**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 standard diluent to each of these two wells and mix thoroughly. Next, transfer 100 μL from both the first and second wells into the third and fourth wells, respectively. Add 50 μL of standard diluent to the third and fourth wells, mix again. Discard 50 μL from each of the third and fourth wells, then add 50 μL to the fifth and sixth wells. Add 50 μL of standard diluent to the fifth and sixth wells and mix. Transfer 50 μL from the fifth and sixth wells into the seventh and eighth wells, then add 50 μL of standard diluent to each and mix. Finally, discard 50 μL from the seventh and eighth wells, and add 50 μL to the ninth and tenth wells. After all dilutions, each well 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, but follow the same procedure otherwise). For the sample wells, add 40 μL of sample diluent, followed by 10 μL of the sample (resulting in a 5-fold dilution). Carefully pipette the sample into the bottom of each well without touching the walls, and gently mix by shaking.
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× or 20×, depending on the kit version) with distilled water as specified in the kit instructions.
5. **Washing**: Remove the sealing film, 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 before proceeding.
6. **Enzyme Reagent Addition**: Add 50 μL of enzyme-labeled reagent to each well except the blank wells.
7. **Second Incubation**: Seal the plate and incubate at 37°C for another 30 minutes.
8. **Second Washing**: Repeat the washing steps as described in point 5.
9. **Color Development**: Add 50 μL of color developer A, followed by 50 μL of color developer B. Gently mix the contents and incubate at 37°C for 15 minutes in the dark.
10. **Stop Reaction**: Add 50 μL of stop solution to each well to terminate the reaction. The color will change from blue to yellow.
11. **Absorbance Measurement**: Measure the absorbance at 450 nm using a microplate reader. Ensure that measurements are taken within 15 minutes after adding the stop solution to avoid interference.
This detailed procedure ensures accurate and reliable quantification of rat cholesterol levels using the Guangrui Bio ELISA Kit. Always follow the manufacturer’s instructions and maintain proper laboratory practices for optimal results.
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