Glycerol Assay Kit, BioAssay™

Référence G8145-10-96T

Conditionnement : 96Tests

Marque : US Biological

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Téléphone : +1 850 650 7790


Shipping Temp
Dry Ice

Storage Temp
4°C/-20°C

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.

Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.