Description
Aspartate Assay Kit (BA0078) (BA0078)
The Aspartate Assay Kit (SKU: BA0078) provides a simple, direct and automation-ready procedure for measuring aspartate concentration. Aspartate, a nonessential amino acid, is a precursor to several other amino acids and an excitatory neurotransmitter, and is involved in the urea cycle and gluconeogenesis. In this assay aspartate is converted into pyruvate, which is then oxidised with the conversion of a dye into a coloured and fluorescent form. The colour intensity at 570 nm, or fluorescence intensity at excitation/emission 530/585 nm, is directly proportional to the aspartate concentration in the sample.
| Product Name: | Aspartate Assay Kit (BA0078) |
| SKU: | BA0078 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 2 to 400 uM; fluorometric 1 to 50 uM aspartate |
| Sample Type: | Plasma, serum, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at 37C or 60 minutes at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of L-aspartate. Aspartate is converted into pyruvate, which is oxidised to convert a dye to a coloured form read at 570 nm or fluorometrically at excitation/emission 530/585 nm.
- Sensitive and accurate
- Colorimetric linear detection range 2 to 400 uM aspartate
- Fluorometric linear detection range 1 to 50 uM aspartate
- Direct and automation-ready procedure
- Direct assays of aspartate in plasma, serum, tissue and culture media
- Drug discovery and pharmacology: effects of drugs on aspartate metabolism
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. Homogenise tissue or cell samples (2 x 10^6) in 100 uL PBS, centrifuge at 14,000 rpm for 5 minutes and use clear supernatant. Deproteinate serum and plasma using a 10 kDa spin filter and use an internal standard; the fluorescent assay is recommended for these low-concentration samples. Avoid culture media with high pyruvate concentrations. |
| 2 | Standards. Dilute the Aspartate Standard to 400 uM by mixing 10 uL 10 mM Standard with 240 uL distilled water, then prepare the dilution series shown in the table. Transfer 25 uL of each standard to separate wells of a clear flat-bottom 96-well plate. |
| 3 | Samples. Add 25 uL of each sample to two separate wells (each sample requires a Sample Blank). For samples needing an internal standard, prepare three reactions (sample plus standard, sample alone, sample blank) using 5 uL 100 uM aspartate where required. |
| 4 | Aspartate detection. Prepare working reagent by combining, for each reaction, 85 uL Developer, 2 uL AST Enzyme, 1 uL ODC Enzyme, 5 uL Cosubstrate and 1 uL Dye Reagent (omit AST Enzyme for Sample Blanks). Add 75 uL of the appropriate working reagent to each well, mix and incubate protected from light for 30 minutes at 37C or 60 minutes at room temperature. Read OD at 570 nm. |
| 5 | Fluorimetric procedure. Dilute the standards 1:10 in distilled water, transfer 25 uL standards and 25 uL samples into a black 96-well plate, add 75 uL working reagent, tap to mix, incubate 30 minutes at 37C or 60 minutes at room temperature and read fluorescence at excitation/emission 530/585 nm. |
Subtract the blank value (standard #4) from the standard values and plot the change in OD or fluorescence against standard concentrations to determine the slope. [Aspartate] = (Rsample - Rblank) / slope x n, where n is the sample dilution factor. When an internal standard is used, [Aspartate] = (Rsample - Rblank) / (Rstandard - Rsample) x 20 uM. If the concentration exceeds the linear range, dilute and repeat. Conversion: 100 uM aspartate equals 13.2 mg/L.
| Component | Quantity | Storage |
| Developer | 10 mL | -20C |
| Dye Reagent | 120 uL | -20C |
| ODC Enzyme | 120 uL | -20C |
| AST Enzyme | 240 uL | -20C |
| Cosubstrate | 600 uL | -20C |
| Aspartate Standard | 400 uL | -20C |
