Choline and its metabolites play important roles in membrane structure integrity, cellular signaling and cholinergic neurotransmission. Aberrant regulation in choline metabolism has been associated with mental illness such as anxiety. This method provides a simple, direct and high-throughput assay for measuring choline in biological samples. In this assay, free choline is oxidized by choline oxidase to betaine and H2O2 which reacts with a specific dye to form a pink colored product. The color intensity at 570nm or fluorescence intensity (530/585 nm) is directly proportional to the choline concentration in the sample.
Key Features:Use 20ul samples. Linear detection range: Colorimetric Assay: 1 to 100uM, fluorometric assay 0.2 to 10uM choline.
Applications:Assays: choline in biological samples such as serum, plasma, urine, saliva, milk, tissue, and cell culture
Drug Discovery/Pharmacology: effects of drugs on choline metabolism.
Kit Contents:Assay Buffer: 10ml Enzyme Mix: 120ulDye Reagent: 120ul Standard: 400ul 2mM CholineStorage 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:Sample treatment: liquid samples such as serum and plasma can be assayed directly. Tissue and cell lysates can be prepared by homogenization in cold 1 x PBS and centrifugation (5 min at 14,000rpm). Use clear supernatants for assay. Milk samples should be cleared by mixing 600ul milk with 100ul 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300ul supernatant into a clean tube and neutralize with 50ul 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n=1.36)
Note: (1). SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol, > 5uM) are known to interfere in this assay and should be avoided in sample preparation. (2). This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough
1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay
2. Standards: mix 12ul 2mM Standard with 228ul dH2O (final 100uM). Dilute standard in dH2O as follows
No 100uM STD+H2O Vol (ul) Choline (uM)1 100ul+0ul 100 1002 60ul+40ul 100 603 30ul+70ul 100 304 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
3. Color reaction. Prepare enough Working Reagent by mixing, for each reaction well, 85ul Assay Buffer, 1ul Enzyme Mix and 1ul Dye Reagent. Add 80ul Working Reagent to each well. Tap plate to mix. Incubate 20 min at room temperature
4. Read optical density at 570nm (550-585nm).
Fluorometric Assay:The Fluorometric Assay: is 10 times more sensitive than the colorimetric method. The procedure is similar to that for the Colorimetric Assay: except that (1) 0, 3, 6 and 10uM choline 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 choline concentration of a sample is higher than 100uM in the Colorimetric Assay: or 10uM in the Fluorometric Assay:, dilute sample in water 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 choline concentration of Sample, [Choline] =RSAMPLE–RBLANKSlope (uM-1)× n (uM)RSAMPLE and RBLANK are optical density or fluorescence intensityreadings of the Sample and H2O Blank, respectively. n is the sampledilution factor
Conversions: 1mM choline equals 10.4 mg/dL, 0.010% or 104 ppm.
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.
