Coenzyme A (CoA) Assay Kit, BioAssay™

Coenzyme A (CoA) is involved in many important biological activities including the synthesis and oxidation of fatty acids, pyruvate oxidation in the citric acid cycle and many others. One of CoA’s most crucial roles is the carrying and transferring of acyl groups. This method provides a simple, two-step and high-throughput assay for measuring CoA. In this assay, the first step enzymatically converts CoA to acyl-CoA and the second step oxidizes the acyl-CoA producing an enoyl-CoA and H2O2. The resulting H2O2 reacts with a specific dye to form a pink colored product. The optical density at 570nm or fluorescence intensity (530/585nm) is directly proportional to the CoA concentration in the sample.||Key Features:|Sensitive. Use 10ul samples. Linear detection range: Colorimetric Assay: 5-1000uM, Fluorometric Assay: 3-100uM CoA.|Convenient. Room temperature “mix-and-read” procedure can be readily automated for high-throughput assay of thousands of samples per day.||Applications:|Assays: CoA in a variety of biological samples.||Kit Contents:|Assay Buffer: 20ml Dye Reagent: 120ul|Enzyme A: 1 2 0ul Enzyme B: 120ul|Substrate: 600ul Standard: 50ul|ATP: 120ul|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. Milk and solid samples can be homogenized in 5% isopropanol and 5% Triton X-100 in water, followed by filtration through a 0.45uM PTFE syringe filter (e.g. VWR Cat# 28145-493).|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. Important: the thawed Standard solution should be clear and colorless. If the Substrate is turbid, bring it to 37°C and gently swirl the tube (do not vortex) until the solution is clear.|2. Standards: Prepare a 1000uM stock of standard by diluting 5ul of the 100mM Standard with 495ul Assay Buffer. Dilute the 1000uM standard in Assay Buffer as follows:|No 1000uM STD+Buffer Vol (ul) Coenzyme A (uM)|1 100ul+0ul 100 1000|2 60ul+40ul 100 600|3 30ul+70ul 100 300|4 0ul +100ul 100 0|Transfer 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. ACS reaction. Prepare enough ACS Working Reagent by mixing, for each well, 40ul Assay Buffer, 1ul Enzyme A, 5ul Substrate, 1ul ATP. Add 40ul ACS WR to each well, tap to mix and incubate at room temperature (RT) for 30 min.|4. ACOD reaction. Prepare enough ACOD Working Reagent by mixing, for each well, 55ul Assay Buffer, 1ul Enzyme B and 1ul Dye Reagent. Add 50ul ACOD Working Reagent to each well. Tap plate to mix. Incubate 30 min at room temperature protected from light.|4. Read optical density at 570nm (550-585nm).||Fluorometric Assay:|The Fluorometric Assay: Procedure: is similar to the Colorimetric Procedure: except that (1) 0, 30, 60 and 100uM Standards 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 (Standard #4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the fatty acid concentration of Sample, |RSAMPLE-RBLANK|Slope|[CoA]=× n (uM)|RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and Blank, respectively. n is the sample dilution factor.|Note: if the calculated CoA concentration of a sample is higher than 1000uM in the Colorimetric Assay: or 100uM in the Fluorometric Assay:, dilute sample in Assay Buffer and repeat the assay. Multiply result by the dilution factor, n.||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. For milk and solid samples, 0.45uM PTFE syringe filter and 5% isopropanol, 5% Triton X-100 solution.