BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at 2-8°C,Protected from light Ships Wet ice Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
High-Density Lipoprotein (HDL), as an anti-atherogenic lipoprotein, transports cholesterol from peripheral tissues to the liver for metabolism, where it is converted into bile acids or directly excreted from the intestine via bile. This process reduces cholesterol deposition on the arterial wall. HDL exerts its anti-atherosclerotic effects through various mechanisms, including promoting reverse cholesterol transport, anti-inflammatory and antioxidant activities, inhibiting thrombus formation, and improving endothelial cell function.
Detection Principle: Cholesterol esterase (CHER) and cholesterol oxidase (CHOD) are chemically modified and used in conjunction with dextran sulfate and magnesium ions (or other compounds like sulfated cyclodextrin complexes) to reduce their enzymatic reactivity towards LDL, VLDL, and chylomicrons, making them selectively interact with HDL-cholesterol. Based on this principle, in the first reaction step, LDL, VLDL, and chylomicrons are complexed with reagents like dextran sulfate. In the second reaction step, using the chemically modified CHER and CHOD, HDL-cholesterol is directly measured without the need to separate other lipoproteins. Specifically, the chemically modified CHER catalyzes the hydrolysis of cholesterol esters to generate Free Cholesterol (FC). FC is then oxidized by CHOD to produce 4-cholestenone and hydrogen peroxide. Subsequently, hydrogen peroxide reacts with 4-aminoantipyrine and phenol under the catalysis of peroxidase (POD) to generate a red quinoneimine compound, which has a characteristic absorption peak at 546 nm. The HDL-C content is determined by measuring the absorbance at 546 nm.
| Component | 96T | Storage |
| Reagent 1 | 18 mL | 2-8℃. Store in the dark. |
| Reagent 2 | 6 mL | 2-8℃. Store in the dark. |
| Reagent 3 | 1EA | 2-8℃. Store in the dark. |
Standard (Powder, 1 vial) Preparation:
1. Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube.
2. Add 0.1 mL of distilled water to dissolve. Use within one week. The prepared concentration is as indicated on the label.
User-Prepared Instruments and Reagents:
Mortar (Homogenizer), balance, ice box (ice maker), benchtop centrifuge, adjustable micropipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water are acceptable), ethanol.
Experimental Procedure
It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue sample and place it in a mortar. Add 1 mL of ethanol and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C or room temperature for 10 minutes. Collect the supernatant for assay.
Note: If increasing the sample amount, maintain a tissue mass (g) to ethanol volume (mL) ratio between 1:5 and 1:10.
1.2 Liquid Samples
Assay clear liquid samples directly. If turbid, centrifuge and use the supernatant for assay.
1.3 Serum Samples
For routine, clear serum samples, add reagents directly according to the assay table and proceed with detection. If the serum sample has a high protein content, adding reagents as per the table may cause turbidity. In this case, first take 200 μL of serum + 200 μL of ethanol, mix well by inverting several times, centrifuge at 8,000 rpm, 4°C or room temperature for 5 minutes, and then collect the supernatant for assay.
1.4 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of ethanol per approximately 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
*Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to ethanol volume (mL) ratio between 500:1 and 1000:1.*
2. Assay Steps
2.1 Preheat the microplate reader for 30 minutes (or wait for the instrument to complete its self-check). Set the wavelength to 546 nm.
2.2 Thaw all reagents to room temperature (25°C). Add reagents sequentially to a 96-well plate as follows:
| Reagent (μL) | Test Tube | Standard Tube (once) | Blank Tube (once) |
| Sample | 2.5 | ||
| Standard | 2.5 | ||
| Distilled Water | 2.5 | ||
| Reagent 1 | 180 | 180 | 180 |
Mix well and incubate at 37°C for 5 minutes. Read the absorbance at 546 nm for each tube (A₁).
| Reagent 2 | 60 | 60 | 60 |
Mix well and incubate at 37°C for 10 minutes. Read the absorbance at 546 nm for each tube (A₂). Calculate ΔA = A₂ - A₁ for each tube.
Note:
(1) If the A₂ value for the Test Tube is greater than 1, dilute the sample with ethanol. The dilution factor (D) must be substituted into the calculation formula.
(2) If ΔA for the Test Tube is lower than ΔA for the Blank Tube, consider increasing the sample volume V₁ (e.g., increase the sample volume in the Test Tube and the water volume in the Blank Tube to 5 μL or more, keeping Reagents 1 and 2 volumes unchanged; for the Standard Tube, keep at 2.5 μL and add 2.5 μL distilled water to make up volume) or increasing the sample weight W (e.g., to 0.2 g or more). The changed V₁ or W must then be substituted into the calculation formula.
3. Calculation of Results
3.1 Based on Sample Mass
Derived Formula:
HDL-C (μmol/g weight) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (W × V₁ ÷ V) × D
Simplified Formula:
HDL-C (μmol/g weight) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ W × D
3.2 Based on Protein Content
Derived Formula:
HDL-C (μmol/mg prot) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (Cpr × V₁ ÷ V) × D
Simplified Formula:
HDL-C (μmol/mg prot) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ Cpr × D
3.3 HDL-C Content in Liquids
Derived Formula:
HDL-C (mmol/L) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ V₁ × D
Simplified Formula:
HDL-C (mmol/L) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × D
3.4 HDL-C Content in Serum
Derived Formula:
HDL-C (mmol/L) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ V₁ × 2 × D
Simplified Formula:
HDL-C (mmol/L) = 2 × CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × D
3.5 Based on Cell Count
Derived Formula:
HDL-C (nmol/10⁴ cells) = (CStandard × V₂) × 10³ × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (500 × V₁ ÷ V) × D
Simplified Formula:
HDL-C (nmol/10⁴ cells) = 2 × CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × D
Parameter Definitions:
CStandard: Concentration as indicated on the label (mmol/L or μmol/mL)
V₁: Volume of sample added (0.0025 mL)
V: Volume of extraction buffer (ethanol) added (1 mL)
V₂: Volume of standard added (0.0025 mL)
D: Dilution factor (1 if not diluted)
2: Dilution factor in serum pre-treatment
500: Number of cells (in units of 10⁴)
W: Sample weight (g)
Cpr: Protein concentration of the supernatant (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
Precautions
1. It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.
2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
Comprehensive hazard, handling, storage, and regulatory compliance document.
Download SDS →Lot-specific quality data. Enter your lot number to retrieve the exact COA.
Look up COA →Full quality attributes and acceptance criteria for this grade.
View spec sheet →Our grade selection guide covers purity, stabilizer status, and application suitability for all variants in our catalog.
View BioReagent grade guide →
