1. Separation of HDL and LDL/VLDL: Mix 100 µL of 2X Precipitation Buffer with 100 µL of serum sample in microcentrifuge tubes. Incubate 10 min at room temperature, centrifuge at 2000 x g (5000 rpm on bench-top microcentrifuge) for 10 min. Transfer the supernatant into new labeled tubes. This is the HDL fraction. The precipitates are the LDL/VLDL fraction. If you want to measure the LDL/VLDL level, the precipitate should be spun again, and the trace amount of HDL supernatant carefully removed. Resuspend the precipitate in 200 µL PBS (not provided). This is the LDL/VLDL fraction. Note A: If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS, and repeat the separation procedure. Multiply final results by two (2) due to the dilution with the 2X Precipitation Buffer. Note B: Precipitation time and temperature do not affect the results significantly.
2. Standard Curve and Sample Preparations: D ilute the Cholesterol Standard to 0.25 μg/µL by adding 20 µL of the Cholesterol Standard to 140 µL of Cholesterol Assay Buffer, mix well. Add 0, 4, 8, 12, 16, 20 µL into a series of wells in a 96-well plate. Adjust volume to 50 µL/well with Cholesterol Assay Buffer to generate 0, 1, 2, 3, 4, 5 μg/well of the Cholesterol Standard. (Note: F luorometric assay is approx. 10 times more sensitive than the colorimetric assay, the cholesterol standards should be diluted 10 times further if using fluorometric assay). For sample testing, using 1 to 20 µL of the HDL or LDL/VLDL fraction, adjust the total volume to 50 µL/well with Cholesterol Assay Buffer. For unknown samples, we suggest testing different volumes of sample to ensure the readings are within the linear portion of the standard curve.
3. Reaction Mix Preparation: Mix enough reagent for the number of assays performed. For each assay, prepare a total 50 µL Reaction Mix containing: 44 µL Cholesterol Assay Buffer 2 µL Cholesterol Probe 1 2 µL Enzyme Mix 2 µL Cholesterol Esterase 2,3 *Notes:
1. If using fluorometric assay, use 0.4 ul of Cholesterol Probe per reaction to reduce fluorescence background.
2. Cholesterol Esterase hydrolyzes cholesteryl ester to free cholesterol. If you want to detect free cholesterol only, omit the Cholesterol Esterase in the reaction, and substitute with 2 µL of Assay Buffer. With the addition of Cholesterol Esterase, the assay detects total cholesterol (cholesterol and cholesteryl esters). If you want to detect cholesteryl esters only, subtract the value of free cholesterol from the value of total cholesterol.
3. Cholesterol Esterase must be added to the reaction for standard curve to convert all the cholesterol in the standard solution.
4. Add 50 µL of the Reaction Mix to each well containing the Cholesterol Standard or test samples, mix well.
5. Incubate the reaction for 60 minutes at 37 °C, protect from light. Measure O.D. at 570 nm or fluorescence at Ex/Em 538/587 nm in a micro-titer plate reader.
Calculation of Results
Calculations: Subtract 0 standard reading from readings. Plot the standard curve. Apply the sample readings to the standard curve to determine sample cholesterol amount in the reaction well. Sample cholesterol concentrations: C = A/V (μg/µL) Where: A is the sample cholesterol amount from the standard curve (in µg). V is original sample volume added to the sample reaction well (in µL). Cholesterol Molecular Weight: 386.6, 1 μg/µL = 100 mg/dL., 2 0.48 0.75 0.23 0.54 1.4 0.23 0.18 0.42 0 0.5 1 1.5 2 Human Serum Rabbit Serum Mouse Serum Total CholesterolHDLLDL/VLDL Measurement of total cholesterol, HDL, LDL/VLDL from serum samples. Total Cholesterol (blue bar), HDL (green bar), and LDL/VLDL (yellow bar) cholesterol were measured following the kit protocol. C holes te rol C onc en trat ion in Serum (µg /µL )