Glycerol [Glycerin or Glycerine, C3H5(OH)3] is widely used in foods, beverages and pharmaceutical formulations. It is also a main by- product of biodiesel production. Simple, direct and automation-ready procedures for measuring glycerol concentrations find wide Applications:. Glycerol assay uses a single Working Reagent that combines glycerol kinase, glycerol phosphate oxidase and color reactions in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to glycerol concentration in the sample.||Key Features:|Sensitive and accurate. Use as little as 10ul samples. Linear detection range in 96-well plate: 10 to 1000uM (92 ug/dL to 9.2 mg/dL) glycerol for Colorimetric Assay:s and 2 to 50uM for Fluorometric Assay:s.|Simple and convenient. The procedure involves addition of a single working reagent and incubation for 20 min at room temperature, compatible for HTS assays.|Improved reagent stability. The optimized formulation has greatly enhanced the reagent and signal stability.||Applications:|Direct Assays: glycerol in biological samples (e.g. serum and plasma).|Drug Discovery/Pharmacology: effects of drugs on glycerol metabolism.|Food and Beverages: glycerol in food, beverages, pharmaceutical formulations etc.||Kit Contents:|Assay Buffer: 24ml Enzyme Mix: 500ul ATP: 250ul|Dye Reagent: 220ul Standard: 100ul 100mM Glycerol|Storage conditions. The kit is shipped on dry ice. Store Assay Buffer at 4°C and other reagents at -20°C. Shelf life of 6 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 Procedure:|Note: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation.|1. Equilibrate all components to room temperature. Keep thawed Enzyme Mix in a refrigerator or on ice. Dilute standard in distilled water as follows (diluted standards can be used for future assays when stored refrigerated).|No STD+H2O Vol (ul) Glycerol (mM)|1 10ul+990ul 1000 1.0|2 6ul+994ul 1000 0.6|3 3ul+997ul 1000 0.3|4 0ul+1000ul 1000 0|Transfer 10ul standards and 10ul samples into separate wells of a clear 96-well plate.|2. For each reaction well, mix 100ul Assay Buffer, 2ul Enzyme Mix, 1ul ATP and 1ul Dye Reagent in a clean tube. This Working Reagent should be used on the same day of preparation. Transfer 100ul Working Reagent into each reaction well. Tap plate to mix.|3. Incubate 20 min at room temperature. Read optical density at 570nm (550-585nm). Note: if the Sample OD is higher than the Standard OD at 1.0mM, dilute sample in water and repeat the assay. Multiply result by the dilution factor.||Calculation:|Subtract blank OD (water, #4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The glycerol concentration of Sample is calculated as|[Glycerol]=ODSAMPLE–ODH2O|Slope|(mM)|ODSAMPLE and ODH2O are optical density values of the sample and water. Conversions: 1mM glycerol equals 9.2 mg/dL, 92 ppm.||Fluorometric Procedure:|For Fluorometric Assays, the linear detection range is 2 to 50uM glycerol. Mix 10ul 100mM Standard with 990ul H2O (final 1mM). No 1mM STD+H2O Vol (ul) Glycerol (mM)|1 50ul+950ul 1000 0.050|2 30ul+970ul 1000 0.030|3 15ul+985ul 1000 0.015|4 0ul +1000ul 1000 0|Dilute standards as above. Transfer 10ul standards and 10ul samples into separate wells of a black 96-well plate. Add 100ul Working Reagent (see Colorimetric Procedure:). Tap plate to mix. Incubate 20 min at room temperature and read fluorescence at lex=530nm and lem=585nm. The glycerol concentration of Sample is calculated as|[Glycerol] =|FSAMPLE–FH2O|Slope|(mM)||Materials Required, But Not Provided:|Pipeting devices, centrifuge tubes, Clear flat-bottom 96-well plates, black 96-well plates (e.g. Corning Costar) and plate reader.