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Protocol
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1. Standard Curve Preparation: For the colorimetric assay, add 10 μ l of the glycerol standard to 990 μ l of Assay Buffer to generate 1 mM glycerol standard, mix well. Add 0, 2, 4, 6, 8, 10 μ l into each well individually. Adjust volume to 50 μ l/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of glycerol Standard. For the fluorometric assay, dilute the Glycerol Standard to 0.01- 0.1 mM with the Assay Buffer (Detection sensitivity is 10-100 fold higher for a fluorometric than a colorimetric assay). Follow the protocol as for the colorimetric assay. 2. Sample Preparation: Prepare test samples to a final volume of 50 μ l/well with Assay Buffer in a 96-well plate. We suggest testing several dilutions of your sample to make sure the readings are within the standard curve range. 3. Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 μ l Reaction Mix: 46 μ l Assay Buffer 2 μ l Glycerol Probe 2 μ l Glycerol Enzyme Mix 4. Add 50 μ l of the Reaction Mix to each well containing standard and samples. Mix well. Incubate at room temperature for 30 minutes, protect from light. 5. Measure O.D. 570 nm for the colorimetric assay or Ex/Em = 535/590 nm for the fluorometric assay in a microtiter plate reader. The reaction is stable for at least two hours. 6. Calculations: Correct background by subtracting the value derived from the 0 glycerol standard from all sample readings. Plot the standard curve (OD 570nm or Fluorescence readings vs. nmol). Apply sample readings to the standard curve. Glycerol concentration can then be calculated: C = Ga / Sv nmol/ μ l or μ mol/ml or mM Where: Ga is Glycerol amount from standard curve (nmol). Sv is the sample volume (before dilution) added in sample wells ( μ l). Glycerol molecular weight: 92.09. VI.
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