Description
Phenylalanine Assay Kit (Fluorometric) (BA0142) (BA0142)
The Phenylalanine Assay Kit (SKU: BA0142) offers a convenient fluorometric means of measuring L-phenylalanine in biological samples. L-phenylalanine is one of the twenty common amino acids and an important precursor of key signalling molecules such as dopamine, noradrenaline, adrenaline and the skin pigment melanin. It occurs naturally in the breast milk of mammals and is used as a nutritional supplement in food and drink products. In this assay L-phenylalanine is oxidised by phenylalanine dehydrogenase, producing NADH, which in turn reduces a fluorescent dye to a highly fluorescent product. The resulting fluorescence intensity, measured at an excitation/emission of 530/585 nm, is proportional to the L-phenylalanine concentration in the sample. The kit provides a sensitive and accurate method suitable for high-throughput automation.
| Product Name: | Phenylalanine Assay Kit (Fluorometric) (BA0142) |
| SKU: | BA0142 |
| Detection Method: | Fluorometric |
| Detection Range: | 2 - 300 uM L-phenylalanine |
| Sample Type: | Serum, urine and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at -20 C except for reconstituted Enzyme A, which is stored at 4 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This kit provides a simple, sensitive fluorometric procedure for the quantitative determination of L-phenylalanine in biological samples. L-phenylalanine is oxidised by phenylalanine dehydrogenase to generate NADH, which reduces a fluorescent dye to a highly fluorescent product measured at 530/585 nm. The homogeneous mix-incubate-measure format requires no wash or transfer steps and can be readily automated for high-throughput screening.
- Safe, non-radioactive assay
- Sensitive and accurate, with a linear detection range of 2 - 300 uM L-phenylalanine
- Convenient homogeneous mix-incubate-measure format with no wash or transfer steps
- High-throughput and readily automated on HTS liquid-handling systems
- Determination of L-phenylalanine in serum, urine and other biological samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reconstitute Enzyme A by adding 120 uL Assay Buffer and mixing thoroughly. Store reconstituted Enzyme A at 4 C (do not freeze) and use within one month. |
| 2 | Prepare samples: homogenise tissue (20 mg) or cells (2x10^6) in 200 uL ice-cold PBS and centrifuge at 14,000 rpm for 5 min. Use the clear supernatant. Store unmeasured samples frozen, preferably at -80 C. |
| 3 | Prepare standards: make 400 uL of 300 uM L-phenylalanine premix by mixing 6 uL 20 mM Standard with 394 uL water, then dilute to 300, 200, 100 and 0 uM. Transfer 10 uL of each standard into separate wells of a black flat-bottom plate. |
| 4 | Transfer 10 uL of each sample in duplicate (Sample and Sample Blank) into separate wells. |
| 5 | Prepare Working Reagent per well by mixing 85 uL Assay Buffer, 8 uL NAD, 5 uL Probe, 1 uL reconstituted Enzyme A and 1 uL Enzyme B. Prepare Blank Reagent per Sample Blank well by mixing 86 uL Assay Buffer, 8 uL NAD, 5 uL Probe and 1 uL Enzyme B (no Enzyme A). |
| 6 | Add 90 uL Working Reagent to standard and sample wells, and 90 uL Blank Reagent to Sample Blank wells. Tap to mix and incubate for 20 min in the dark. |
| 7 | Read fluorescence intensity at an excitation/emission of 530/585 nm. |
| 8 | For complex matrices such as serum or urine, follow the internal standard (kinetic) method with reading of initial rates. |
Plot the L-phenylalanine standard curve and determine its slope. Calculate the sample concentration as [L-Phenylalanine] = (F_SAMPLE - F_BLANK) / Slope (uM). If the concentration exceeds 300 uM, dilute the sample in water, repeat the assay and multiply by the dilution factor. One uM L-phenylalanine is equivalent to 165 ug/L or 165 ppb.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| NAD Solution | 1 mL | -20 C |
| Probe | 750 uL | -20 C |
| Enzyme A | Dried | -20 C (4 C once reconstituted) |
| Enzyme B | 120 uL | -20 C |
| Standard | 120 uL | -20 C |
