Phenylalanine Assay Kit

Details for Product No. ABIN1000326, Supplier: Log in to see
Detection Range
2-300 μM
Minimum Detection Limit
2 μM
Biochemical Assay (BCA)
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Supplier Product No.
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Sample Type Serum, Urine
Characteristics Safe. Non-radioactive assay.
Sensitive and accurate. Linear detection range of 2 - 300 µM L- phenylalanine.
Convenient and high-throughput.
Homogeneous mix-incubate-measure type assay. No wash and reagent transfer steps are involved. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
Components Assay Buffer: 10 mL. Enzyme A: 120 µL. NAD Solution: 1 mL. Enzyme B: 120 µL. Probe: 120 µL. Standard: 120 µL.
Material not included Pipetting devices, centrifuge tubes, black flat bottom 96-well plates and plate reader.
Background Quantitative determination of L-phenylalanine by fluorimetric (530/595nm) method.
Procedure: 30 min.

L-Phenylalanine is one of the twenty common amino acids and an important precursor for several key signal molecules such as dopamine, norepinephrine, epinephrine, and the skin pigment melanin. It is found naturally in the breast milk of mammals, and used as nutritional supplements in food and drink products. The genetic disorder phenylketonuria is the inability to metabolize phenylalanine. Individuals with this disorder are known as phenylketonurics. Individuals who cannot metabolize phenylalanine must monitor their intake of protein to control the buildup of phenylalanine. This L-Phenylalanine Assay Kit provides a convenient fluorimetric means to measure L-phenylalanine in biological samples. In the assay, L-phenylalanine is oxidized by phenylalanine dehydrogenase, producing NADH, which reduces a fluorescent dye to a highly fluorescent product. The resulting fluorescence intensity (exc/em = 530/595nm) is linear to the L-phenylalanine concentration in the sample.
Application Notes Determination of L-phenylalanine in serum, urine and other biological samples.
Protocol Use black flat-bottom plates. Prior to assay, bring all reagents to room temperature. Briefly centrifuge enzyme tubes, keep on ice during assay.
1. Standards. Prepare 400 µL 300 µML-Phenylalanine Premix by mixing 6 µL 20 mM Standard and 394 µL distilled water. Transfer 5 µL standards into separate wells of the plate.
2. Sample. Liquid samples can be assayed directly. Tissue (20 mg) or cells (2x10 6 ) can be homogenized in 200 µL ice-cold PBS, followed by centrifugation at 14,000 rpm for 5 min. Use clear supernatant for assay. Samples not measured on the same day can be stored frozen, preferably at -80°C. Transfer 5 µL of each sample in duplicate, one for Sample and one for Sample Blank, to separate wells of the plate.
3. Assay. For standards and sample wells, prepare enough Working Reagent, for each well, by mixing 85 µL Reagent A, 8 µL NAD, 1 µL Probe, 1 µL Enzyme A and 1 µL Enzyme B. For the Sample Blank wells, prepare Blank Reagent for each well by mixing 86 µL Reagent A, 8 µL NAD, 1 µL Probe and 1 µL Enzyme B (i.e., without Enzyme A). Add 90 µL Working Reagent to Standard and Sample wells, and 90 µL Blank Reagent to the Sample Blank wells. Tap plate to mix. Incubate for 20 min in the dark.
5. Read fluorescence intensity at exc/em = 530/595 nm.
Calculation of Results

Plot the L-phenylalanine Standard Curve and determine its Slope.
Conversion factor: 1 µM L-phenylalanine is equivalent to 165 µg/L or 165 ppb.

Restrictions For Research Use only
Storage -20 °C
Supplier Images
Biochemical Assay (BCA) image for Phenylalanine Assay Kit (ABIN1000326) Phenylalanine Assay Kit
Background publications Campbell, Hollifield, Varsani, Milligan, Brearley, Price: "Development of an enzyme-mediated assay for phenylalanine in blood spots." in: Annals of clinical biochemistry, Vol. 31 ( Pt 2), pp. 140-6, 1994 (PubMed).

Mehrle, DeClue: "Phenylalanine determination in fish serum: adaptation of a mammalian method to fish." in: Analytical biochemistry, Vol. 52, Issue 2, pp. 660-1, 1973 (PubMed).

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