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. This 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 20ul samples. Linear detection range: Colorimetric Assay: 3-200uM Fluorometric Assay: 0.6-20uM 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.
Storage and Stability:Store at -20°C. Stable for 3 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
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, > 5uM), 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 24ul 2mM Standard with 216ul dH2O (final 200uM). Dilute standard in dH2O as follows
No 200uM STD+H2O Vol (ul) Standard (uM)1 100ul+0ul 100 2002 60ul+40ul 100 1203 30ul+70ul 100 604 0ul +100ul 100 0Transfer 20ul diluted standards into separate wells of a clear flatbottom 96-well plate
Samples: transfer 20ul 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 20ul of the sample
3. Color reaction. Prepare enough Working Reagent by mixing, for each well, 85ul Assay Buffer, 1ul PLD Enzyme, 1ul Enzyme Mix and 1ul Dye Reagent. Add 80ul Working Reagent to each well. For samples that contain choline, prepare a blank control reagent with no PLD Enzyme (i.e., 85ul Assay Buffer, 1ul Enzyme Mix and 1ul Dye Reagent). Add 80ul 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).
Fluorometric Assay:The Fluorometric Assay: Procedure: is similar to the Colorimetric Procedure: except that (1) 0, 6, 12 and 20uM 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 200uM in the Colorimetric Assay: or 20uM in the Fluorometric 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, [Phospholipid] =RSAMPLE–RBLANKSlope (uM-1)× n (uM)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.
