磷脂测试盒
产品名称： 磷脂测试盒
英文名称： 'EnzyChrom™ Phospholipid Assay Kit
产品编号： EPLP-048， EPLP-100
产品价格： 0
产品产地： 美国
品牌商标： BioAssay Systems（美国博世）
更新时间： null
使用范围： null
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Description Phospholipids are a class of lipids which constitute a major component of cell membranes and play important roles in signal transduction. Most phospholipids contain one diglyceride, a phosphate group, and one choline. BioAssay Systems method provides a simple, direct and high-throughput assay for measuring choline-containing phospholipids in biological samples. In this assay, phospholipids (such as lecithin, lysolecithin and sphingomyelin) are enzymatically hydrolyzed to choline which is determined using choline oxidase and a H2O2 specific dye. The optical density of the pink colored product at 570nm or fluorescence intensity (530/585 nm) is directly proportional to the phospholipid concentration in the sample.
Key features Sensitive. Use 20 mL samples. Linear detection range: colorimetric assay 3 - 200 mM, fluorimetric assay 0.6 - 20 mM phospholipid.
Applications Assays: phospholipid in biological samples such as serum and non-EDTA plasma. Drug Discovery/Pharmacology: effects of drugs on choline-containing phospholipid metabolism.
Kit Contents Assay Buffer: 10 mL PLD Enzyme: 120 mL Enzyme Mix: 120 mL Dye Reagent: 120 mL Standard: 400 mL 2 mM phosphatidylcholine Storage conditions. The kit is shipped on ice. Store all components at -20°C. Shelf life of three 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.
Colorimetric Assay Liquid samples such as serum and plasma can be assayed directly. Solid samples can be homogenized in the assay buffer. Note: SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol, > 5 mM), sodium azide, EDTA, and sodium dodecyl sulfate are known to interfere in this assay and should be avoided in sample preparation.
1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay. 2. Standards: mix 24 mL 2 mM Standard with 216 mL dH2O (final 200 mM). Dilute standard in dH2O as follows. No 200 mM STD + H2O Vol (mL) Standard (mM) 1 100 mL + 0 mL 100 200 2 60 mL + 40 mL 100 120 3 30 mL + 70 mL 100 60 4 0 mL +100 mL 100 0 Transfer 20 mL diluted standards into separate wells of a clear flat-bottom 96-well plate. Samples: transfer 20 mL of each sample into separate wells of the plate. Note: if a sample is known to contain choline, prepare an extra sample blank well with 20 mL of the sample. 3. Color reaction. Prepare enough Working Reagent by mixing, for each well, 85 mL Assay Buffer, 1 mL PLD Enzyme, 1 mL Enzyme Mix and 1 mL Dye Reagent. Add 80 mL Working Reagent to each well.
For samples that contain choline, prepare a blank control reagent with no PLD Enzyme (i.e., 85 mL Assay Buffer, 1 mL Enzyme Mix and 1 mL Dye Reagent). Add 80 mL of the Control Reagent to the Sample Blank well.
Tap plate to mix. Incubate 30 min at room temperature. Note: if precipitation occurs with certain samples, carry out the reaction in centrifuge tubes. After the 30 min incubation, centrifuge 5 min at 14,000 rpm. Transfer the supernatant into the wells for OD reading. 4. Read optical density at 570nm (550-585nm).
Fluorimetric Assay The fluorimetric assay procedure is similar to the colorimetric procedure except that (1) 0, 6, 12 and 20 mM phospholipid standards and (2) a black 96-well plate are used. Read fluorescence intensity at lex = 530 nm and l em = 585 nm.
Note: if the calculated phospholipid concentration of a sample is higher than 200 mM in the Colorimetric Assay or 20 mM in the Fluorimetric Assay, dilute sample in 0.5% Triton X-100 and repeat the assay. Multiply result by the dilution factor n.
CALCULATION Subtract blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the phospholipid concentration of Sample, RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and H2O Blank (or Sample Blank if sample contains choline), respectively. n is the sample dilution factor.
Materials Required, but not provided Pipetting devices, centrifuge tubes, clear flat-bottom uncoated 96-well plates, optical density plate reader; black flat-bottom uncoated 96-well plates, fluorescence plate reader. 96-well colorimetric assay 96-well fluorimetric assay Literature
1. Nie, Y. et al. (1993). A micro enzymic method for determin