Phospholipase D (PLD) catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. Abnormalities in PLD expression have been associated with human cancers. This method provides a simple and high-throughput assay for measuring PLD activity. In this assay, PLD hydrolyzes phosphatidylcholine 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 PLD activity in the sample.
Simple and High-throughput: the assay involves addition of a single working reagent and can be readily adapted to high-throughput assays for drug screening.
Applications:Direct Assays: phospholipase D in biological samples
Drug Discovery/Pharmacology: effects of drugs on phospholipase D metabolism.
Kit Contents:Assay Buffer: 10ml Dye Reagent: 120ulEnzyme Mix: 120ul Substrate: 1.5mlCalibrator: 400ulStorage 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 can be assayed directly. Solid samples should be homogenized in a suitable enzyme buffer prior to assay
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. If a sample is known to contain choline, it should be removed by dialysis or membrane filtration
1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay
2. Calibrator: mix 33ul Calibrator with 187ul dH2O (final 300uM choline). Dilute calibrator in dH2O as follows
No 300uM Premix+H2O Vol (ul) Calibrator (uM)1 100ul+0ul 100 3002 60ul+40ul 100 1803 30ul+70ul 100 904 0ul +100ul 100 0Transfer 10ul diluted standards into separate wells of a clear flatbottom 96-well plate. Samples: transfer 10ul of each sample into separate wells of the plate
3. Color reaction. Prepare enough Working Reagent by mixing, for each well, 85ul Assay Buffer, 1ul Enzyme Mix, 1ul Dye Reagent and 12ul Substrate. Add 90ul Working Reagent to each well. Tap plate to mix. Incubate at desired temperature and protect from light. At 10 and 30 min, read optical density 570nm (550-585nm).
Fluorometric Assay:The Fluorometric Assay: Procedure: is similar to the Colorimetric Procedure: except that (1) 0, 9, 18 and 30uM calibrator and (2) a black 96-well plate are used. Read fluorescence intensity at lex=530 nm and l em=585 nm.
Calculation:Subtract blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the PLD activity of Sample,[Phospholipase D] =R30–R10Slope x 20× n (U/L)R30 and R10 are optical density or fluorescence intensity readings of the Sample at 30 min and 10 min, respectively. 20 is the enzyme reaction time (30 min-10 min). n is the sample dilution factor. Note: if the calculated PLD activity of a sample is higher than 10 U/L in theColorimetric Assay: or 1 U/L in the Fluorometric Assay:, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor
Unit definition: 1 unit of PLD catalyzes formation of 1uMole of choline per minute under the assay conditions (pH 7.4).
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.
