Phenylalanine Assay Kit, BioAssay(TM)

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.\n\nL-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 (lexc/em=530/595nm) is linear to the L-phenylalanine concentration in the sample.\n\nKey Features:\nSafe. Non-radioactive assay.\nSensitive and accurate. Linear detection range of 2-300uM L-phenylalanine.\nConvenient 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.\n\nApplications:\nDetermination of L-phenylalanine in serum, urine and other biological samples.\n\nKit Contents:\nAssay Buffer: 10ml Enzyme A: 120ul\nNAD Solution: 1ml Enzyme B: 120ul\nProbe: 120ul Standard: 120ul\nStorage conditions: store all reagents at -20 degrees C. Shelf life of 6 months after\nreceipt.\nPrecautions: reagents are for research use only. Normal precautions for\nlaboratory reagents should be exercised while using the reagents. Please\nrefer to Material Safety Data Sheet for detailed information.\n\nAssay Procedure:\nUse black flat-bottom plates. Prior to assay, bring all reagents to room\ntemperature. Briefly centrifuge enzyme tubes, keep on ice during assay.\n1. Standards. Prepare 400ul 300uM L-Phenylalanine Premix by mixing 6\nul 20mM Standard and 394ul distilled water. Dilute standard as\nfollows.\nNo Premix+H2O Standard (uM)\n1 90ul+0ul 300\n2 60ul+30ul 200\n3 30ul+60ul 100\n4 0ul+100ul 0\nTransfer 5ul standards into separate wells of the plate.\n2. Sample. Liquid samples can be assayed directly. Tissue (20 mg) or cells\n(2x106) can be homogenized in 200ul ice-cold PBS, followed by\ncentrifugation at 14,000 rpm for 5 min. Use clear supernatant for assay.\nSamples not measured on the same day can be stored frozen,\npreferably at -80 degrees C.\nTransfer 5ul of each sample in duplicate, one for Sample and one for\nSample Blank, to separate wells of the plate.\n3. Assay. For standards and sample wells, prepare enough Working\nReagent, for each well, by mixing 85ul Reagent A, 8ul NAD, 1ul\nProbe, 1ul Enzyme A and 1ul Enzyme B.\n4. For the Sample Blank wells, prepare Blank Reagent for each well by\nmixing 86ul Reagent A, 8ul NAD, 1ul Probe and 1ul Enzyme B\n(i.e., without Enzyme A).\nAdd 90ul Working Reagent to Standard and Sample wells, and 90ul\nBlank Reagent to the Sample Blank wells. Tap plate to mix. Incubate for\n20 min in the dark.\n5. Read fluorescence intensity at lexc/em=530/595nm.\n\nCalculation:\nPlot the L-phenylalanine Standard Curve and determine its Slope.\nPhenylalanine concentration of a Sample is calculated as\n[L-Phenylalanine] =\nFSAMPLE

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SPECIFICATIONS

Catalog Number

P4060-05G

Size

1Kit

References

1. Campbell RS et al (1994). Development of an enzyme-mediated assay for phenylalanine in blood spots. Ann Clin Biochem 31(2):140-6.\n2. Hummel W et al (1988). Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase. Anal Biochem 170(2):397-401.\n3. Mehrle PM, DeClue ME (1973). Phenylalanine determination in fish serum: adaptation of a mammalian method to fish. Anal Biochem 52(2):660-1.