The samples (including serum) are added as aseprate set of well and are not mixed with the Table 1 standards.
Each well receives 50 µL of sample/standard/blank first, followed by addition of the reaction mix prepared using the reagents listed in Table 2. There will be wells for samples, standards, and a standard blank.
Below see a brief summary:
1. Prepare standard wells and sample wells.
2. Add the appropriate amount of reaction mix.
3. Measure the absorbance (assuming colorimetric detection)
Preparation
Page 2 covers sample preparation first, followed by standard preparation.
Sample Preparation :
Determine an appropriate sample amount. The final volume should be 50 µL.
For example, assuming 50 µL is the correct sample volume, dispense 50 µL of the sample into a single well of a 96-well plate.
1. Standard Preparation:
Perform an 8-fold dilution of the 2 µg/µL stock to reach 0.25 µg/µL to prepare a 1x standard.
Follow the instructions to prepare 12 standard wells, with a final volume of 50 µL each, using that diluted stock.
2. Prepare the reaction mix:
Determine the amount of reaction mix to prepare. Determine if total or free cholesterol is to be measured.
Table 2 shows how to prepare the reaction mix in a single vial and which wells to add to.
If measuring 13 wells for total cholesterol, multiply each component (assay buffer, probe, enzyme mix, cholesterol esterase) by 13.
3. Begin the assay reaction
Add 50 µL of the prepared reaction mix to each sample and standard well.
Proceed with the remaining steps described in the technical bulletin provided at the link below.
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/129/825/cs0005bul-ms.pdf