Description
D-Amino Acid Assay Kit (BA0237) (BA0237)
D-amino acids are not as widespread as their enantiomeric counterparts in proteins, but they can be found in organisms ranging from bacteria (cell walls and antibiotics) to mammals (central nervous systems). The presence of D-amino acids in food is also of considerable interest, as racemisation of L-amino acids during food processing may affect food quality and nutritional value. The D-Amino Acid Assay Kit (SKU: BA0237) uses an enzyme-catalysed oxidation of D-amino acids to convert a dye into a coloured and fluorescent form. The absorbance at 570 nm or fluorescence intensity at λex/em = 530/585 nm is directly proportional to the D-amino acid concentration in the sample. The single working-reagent format is read after 60 minutes and is readily automated to process thousands of samples per day.
| Product Name: | D-Amino Acid Assay Kit (BA0237) |
| SKU: | BA0237 |
| Detection Method: | Colorimetric (OD570 nm) / Fluorimetric (λex/em = 530/585 nm) |
| Detection Range: | 0.86 to 500 µM (colorimetric) and 0.18 to 50 µM (fluorimetric) for 60 min reaction |
| Sample Type: | Tissue, milk and other biological preparations |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped on ice. Store all components at -20°C upon receiving. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
A quantitative colorimetric and fluorimetric assay for D-amino acids using an enzyme-catalysed oxidation of D-amino acids to convert a dye into a coloured and fluorescent form. The absorbance at 570 nm or fluorescence intensity at λex/em = 530/585 nm is directly proportional to the D-amino acid concentration in the sample.
- Fast and sensitive. Linear detection range: 0.86 to 500 µM (colorimetric assay) and 0.18 to 50 µM (fluorimetric assay) for 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.
- D-Amino Acid determination in tissue, milk, and other biological preparations.
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 samples can be prepared by homogenising 20-100 mg in 200-1000 µL dH2O, centrifuging at 10,000 × g for 15 min at 4°C and removing supernatant for assay. Milk samples often require at least 2× dilution in Assay Buffer. |
| 2 | Reagent preparation: equilibrate all reagents to room temperature; briefly centrifuge tubes before opening. |
| 3 | Colorimetric 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 tubes (500, 300, 150, 0 µM) per the 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 assay wells by mixing, per well, 85 µL Assay Buffer, 1 µL DAA Enzyme, 1 µL HRP Enzyme and 1 µL Dye Reagent. |
| 6 | Add 80 µL WR to each well. Tap plate briefly to mix. Incubate at room temperature for 60 min. Measure OD570nm. |
| 7 | Fluorimetric procedure: dilute the colorimetric standards 1:10 in dH2O; transfer 20 µL standards and 20 µL samples into separate wells of a black flat-bottom 96-well plate. Add 80 µL WR to each well, tap to mix, incubate protected from light for 60 min at room temperature and measure 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 to determine the slope. [D-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 |
| HRP Enzyme | 120 µL | -20°C |
| DAA Enzyme | 120 µL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Standard | 500 µL | -20°C |
