Lipoprotein, High Density (HDL) and Lipoprotein, Low Density (LDL)/Lipoprotein, Very Low Density (VLDL) BioAssay™ Kit, Colorimetric

Cholesterol concentrations in High-Density Lipoprotein (HDL) and Low-Density (LDL)/Very-Low-Density (VLDL) Lipoproteins are strong predictors for coronary heart disease. Functional HDL offers protection by removing cholesterol from cells and atheroma. Higher concentrations of LDL and lower concentrations of functional HDL are strongly associated with cardiovascular disease due to higher risk of atherosclerosis. The balances between high- and low-density lipoproteins are solely genetically determined, but can be changed by medications, food choices and other factors. ||Simple, direct and automation-ready procedures for measuring HDL and LDL/VLDL concentrations are very desirable. HDL and LDL/VLDL quantification kit is based on our improved PEG precipitation method in which HDL and LDL/VLDL are separated, and cholesterol concentrations are determined using cholesterol esterase/cholesterol dehydrogenase reagent. In this reaction, NAD is reduced to NADH. The optical density of the formed NADH at 340 nm is directly proportionate to the cholesterol concentration in the sample.||Applications:|Direct Assays: HDL and LDL/VLDL cholesterol in serum samples from any species.|Pharmacology: evaluation of drugs on cholesterol metabolism.||Key Features:|Sensitive and accurate. Requires only 20ul serum sample.|Detection limit of 5 mg/dL, linearity up to 300 mg/dL cholesterol in 96-well plate assay.|Convenient. Room temperature assay. No 37°C heater is needed.||Kit Contents (100 assays in 96-well plates):|PBS: 1.5ml Precipitation Reagent: 1.5ml|Assay Buffer: 20ml Enzyme Mix: 120uL|NAD Solution: 2ml Standard: 1ml 300mg/dL cholesterol||Storage conditions:|Store PBS and Precipitation Reagent at room temperature and the rest reagents at -20°C. Shelf life: 6 months after receipt.||Precautions: |Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|Important: bring all reagents except enzyme mix to room temperature prior to assay. Non-hemolyzed serum samples should be used. The following procedure is designed for duplicate determinations.|1. Sample Preparation. Transfer 20ul serum into a 1.5-mL centrifuge tube, add 20ul Precipitation Reagent. Vortex to mix and centrifuge 5 min at 9,500 x g (e.g. 9,500 rpm in an Eppendorf 5415C tabletop centrifuge). Carefully transfer 24ul supernatant into a clean tube, add 96ul Assay Buffer. Label this tube “HDL”. Carefully remove all remaining supernatant from the pellet. Transfer 40ul PBS to the pellet and mix by repeated pipetting. Transfer 24ul mixture into another clean tube, add 96ul Assay Buffer. Label this tube “LDL/VLDL”. In a third tube, transfer 12ul serum sample and mix well with 108ul Assay Buffer. Label this tube “Total”. Cholesterol Standard: transfer 12ul 300 mg/dL cholesterol and mix with 108ul Assay Buffer. Label this tube “Standard”.|2. Assay. Transfer 50ul Assay Buffer (“Blank”), 50ul Standard, 50ul “Total”, 50ul “HDL” and 50ul “LDL/VLDL” into wells of a clear bottom 96-well plate. If desired, run assays in duplicate. Prepare enough Working Reagent. For each reaction well, mix 50ul Assay Buffer, 18ul NAD Solution and 1ul Enzyme Mix. Transfer 60ul of the Working Reagent to each reaction well. Tap plate to mix well. Note: addition of Working Reagent to all wells should be rapid and mixing should be thorough. Use of a multichannel pipettor is recommended. Incubate 30 min at room temperature. Read OD values at 340nm.|3. Calculation:. Cholesterol concentrations in the Total, HDL and (LDL/VLDL) fractions are calculated as follows,|[Total] =|ODTOTAL–ODBLANK|ODSTANDARD–ODBLANK|× 300 (mg/dL)|[HDL] =|ODHDL–ODBLANK|ODSTANDARD–ODBLANK|× 300 (mg/dL)|[LDL/VLDL] =|ODLDL/VLDL–ODBLANK|ODSTANDARD–ODBLANK|× 300 (mg/dL)||Materials Required, But Not Provided:|Pipetting (multi-channel) devices, clear bottom 96-well plate and plate reader.||Examples:|Serum samples were run in duplicate according to the standard procedure.