Description
L-Amino Acid Kit (BA0251) (BA0251)
The L-Amino Acid Kit (SKU: BA0251) offers a fast and sensitive method for the quantitative colorimetric or fluorimetric determination of L-amino acids in biological and food samples. L-amino acids are the building blocks of proteins, with almost all of the common 20 amino acids existing as the L-enantiomer. The assay uses an enzyme-catalysed oxidation of L-amino acids to convert a dye into a coloured and fluorescent form, such that the absorbance at 570 nm or fluorescence intensity at λex/em = 530/585 nm is directly proportional to the L-amino acid concentration in the sample. Linear detection ranges are 3.3 to 500 µM for the colorimetric assay and 0.13 to 50 µM for the fluorimetric assay over a 60-minute reaction. The convenient homogeneous mix-incubate-measure procedure involves adding a single working reagent and reading after 60 minutes, and can be readily automated to process thousands of samples per day.
| Product Name: | L-Amino Acid Kit (BA0251) |
| SKU: | BA0251 |
| Detection Method: | Colorimetric (OD570nm) and fluorimetric (λex/em = 530/585 nm) |
| Detection Range: | 3.3 to 500 µM (colorimetric); 0.13 to 50 µM (fluorimetric) |
| Sample Type: | Serum, culture media, tissue homogenates, cell lysates, urine and food samples |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A quantitative colorimetric and fluorimetric assay for L-amino acids. An enzyme-catalysed oxidation of L-amino acids converts a dye into a coloured and fluorescent form measured at 570 nm (absorbance) or λex/em = 530/585 nm (fluorescence).
- Fast and sensitive. Linear detection range: 3.3 to 500 µM (colorimetric assay) and 0.13 to 50 µM (fluorimetric assay) for a 60 min reaction.
- Convenient. The procedure involves adding a single working reagent and reading after 60 minutes.
- High-throughput. Homogeneous mix-incubate-measure type assay. Can be readily automated to process thousands of samples per day.
- L-amino acid determination in serum, culture media, tissue homogenates, cell lysates, urine, food samples, etc.
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 | Sample preparation. Tissue or solid samples (e.g. food): homogenise 20-100 mg sample in 200-1000 µL dH2O, centrifuge at 10,000 × g for 15 min at 4°C and remove supernatant. Cell lysate: collect cells by centrifugation at 2,000 × g for 5 min at 4°C (use a rubber policeman for adherent cells), homogenise or sonicate in cold buffer containing 50 mM potassium phosphate (pH 7.5), centrifuge at 14,000 × g for 10 min at 4°C and remove supernatant. Liquid samples can be assayed directly; dilute serum and cell culture media 4-fold in dH2O, and for urine use an internal standard method. |
| 2 | Reagent preparation. Equilibrate all reagents to room temperature. |
| 3 | Colorimetric procedure - Standards: prepare 200 µL of 500 µM Premix by mixing 50 µL Standard (2 mM) and 150 µL dH2O. Dilute standards in 1.5-mL centrifuge tubes as in the standards table. Transfer 20 µL standards into separate wells of a clear flat-bottom 96-well plate. |
| 4 | Transfer 20 µL of each sample into separate wells. |
| 5 | Prepare enough Working Reagent (WR) for all wells by mixing, per well: 85 µL Assay Buffer, 1 µL LAA Enzyme, 1 µL HRP Enzyme and 1 µL Dye Reagent. Add 80 µL WR to each well. Tap the plate briefly to mix. |
| 6 | Incubate at room temperature for 60 min and read OD570nm. |
| 7 | Fluorimetric procedure: dilute the standards prepared above 1:10 in dH2O. Transfer 20 µL standards and 20 µL samples into separate wells of a black 96-well plate. Add 80 µL Working Reagent to each well and tap to mix. Incubate protected from light for 60 min at room temperature and read fluorescence at λex/em = 530/585 nm. |
Subtract the blank value (water, standard #4) from the standard values and plot the adjusted values against standard concentrations. Determine the slope and calculate the L-amino acid concentration: [L-Amino Acid] = (R_SAMPLE – R_BLANK) / Slope (µM⁻¹) × n (µM), where R_SAMPLE and R_BLANK are the OD or fluorescence values of the sample and H2O blank respectively, and n is the sample dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20°C |
| LAA Enzyme | 120 µL | -20°C |
| HRP Enzyme | 120 µL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Standard | 1 mL | -20°C |
