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Protokoll
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1. Carnitine Standard Curve: For the Colorimetric Assay: Dilute 10 μl of the 100 mM Carnitine Standard with 990 μl dH2O to generate 1 mM standard Carnitine. Add 0, 2, 4, 6, 8, 10 μl of the diluted Carnitine Standard into a 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well standard. Bring the volume to 50 μl with Assay Buffer. For the Fluorometric Assay: Dilute the standard to 0.1 mM (0.1 nmol/ μl), then follow the same protocol as colorimetric assay. Will give 0, 0.2, 0.4, 0.6, 0.8, 1 nmol/well standard Curve. 2. Sample Preparations: Tissues (10 mg) or cells (1×10 6 ) can be homogenized in 100 μl Assay Buffer, centrifuge to remove insoluble materials at 13,000 g for 10 minutes. 10-50 μl deproteinized serum samples can be directly diluted in the Assay Buffer. Bring sample wells to 50 μl/well with Assay Buffer in a 96-well plate. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. Deproteinization may be done by PCA precipitation followed by KOH neutralization (BioVision, Cat.# K808-200) or by using centrifugation through a 10 kD MW cut-off filter (BioVision, Cat.# 1997-25). The normal range for serum L-Carnitine is ~20-50 μM. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing: L-Carnitine Measurement Background Control* Assay Buffer 40 μl 44 µl Carnitine Converting Enzyme 2 μl ------ Carnitine Development Mix 2 μl 2 μl Carnitine Substrate Mix 4 µl 4 µl Carnitine Probe 2 μl 2 μl ** * Perform background control if high levels of acyl –CoA’s or free Coenzyme A are suspected to be in your samples. Choline in samples will give a positive signal but is present at ~10% of the Carnitine concentration. ** for the fluorescent assay, dilute the probe 10X to reduce background reading. Add 50 μl of the Reaction Mix to each well containing the Carnitine standard and test samples. Mix well. Incubate the reaction for 30 min at room temperature, protect from light. 4. Measure O.D. at 570 nm, or fluorescence at Ex/Em 535/587 nm in a microplate reader. 5. Calculation: Correct background by subtracting the value derived from the 0 Carnitine control from all sample and standard readings (The background reading can be significant and must be subtracted from sample readings). Plot the standard Curve. Apply sample readings to the standard curve to have the amount of Carnitine in sample wells S a . Carnitine concentrations of the test samples can then be calculated: C = S a /S v (nmol/μl, or mM) where S a is the Carnitine content of test samples (in nmol) from standard curve, S v is sample volume (in μl) added into the assay wells. L-Carnitine Molecular Weight is 161.2 g/mol.
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