Description
L-Alanine Assay Kit (Colorimetric/Flurometric) (BA0073) (BA0073)
The L-Alanine Assay Kit (SKU: BA0073) provides a simple, direct and automation-ready procedure for measuring alanine concentration. Alanine, a nonessential amino acid, is utilised in the glucose-alanine cycle between tissues and the liver: alanine transaminase transfers the amine group from glutamate to pyruvate to form alanine and alpha-ketoglutarate, and the alanine is transported to the liver where pyruvate is regenerated and converted to glucose. Alanine concentration may correlate with blood pressure, energy intake, cholesterol levels and body mass index. In this assay alanine is converted into pyruvate, which is then measured directly. The colour intensity at 570 nm, or fluorescence intensity at excitation/emission 530/585 nm, is directly proportional to the alanine concentration.
| Product Name: | L-Alanine Assay Kit (Colorimetric/Flurometric) (BA0073) |
| SKU: | BA0073 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 1 to 200 uM; fluorometric 0.4 to 20 uM alanine |
| Sample Type: | Plasma, serum, urine, 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-alanine. Alanine is converted into pyruvate, which is measured with a dye read at 570 nm or fluorometrically at excitation/emission 530/585 nm.
- Sensitive and accurate
- Colorimetric linear detection range 1 to 200 uM alanine
- Fluorometric linear detection range 0.4 to 20 uM alanine
- Direct and automation-ready procedure
- Direct assays of alanine in plasma, serum, urine, tissue and culture media
- Drug discovery and pharmacology: effects of drugs on alanine 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. Dilute serum at least 10-fold in distilled water or deproteinate using a 10 kDa spin filter. Avoid culture media with high pyruvate concentrations. |
| 2 | Standards. Dilute the Alanine Standard to 200 uM by mixing 5 uL 20 mM Standard with 495 uL distilled water, then prepare the dilution series shown in the table. Transfer 50 uL of each standard to separate wells of a 96-well plate. |
| 3 | Alanine detection. Prepare working reagent by combining, for each reaction, 50 uL Developer, 1 uL ALT Enzyme, 5 uL Cosubstrate and 1 uL Dye Reagent. Add 50 uL working reagent to each standard and sample well, mix and incubate protected from light for 30 minutes at 37C or 60 minutes at room temperature. |
| 4 | Read OD at 570 nm. |
| 5 | Fluorimetric procedure. Dilute the standards 1:10 in distilled water, transfer 50 uL standards and 50 uL samples into a black 96-well plate, add 50 uL working reagent, tap to mix, incubate 30 minutes at 37C or 60 minutes at room temperature and read fluorescence at excitation 530 nm and emission 585 nm. |
Subtract the blank value (standard #4) from the standard values and plot the change in OD or fluorescence against standard concentrations. Determine the slope and calculate [Alanine] = (Rsample - Rblank) / slope x n, where n is the sample dilution factor. If the concentration exceeds the linear range, dilute and repeat. Conversion: 1 mg/dL alanine equals 112.2 uM.
| Component | Quantity | Storage |
| Developer | 6 mL | -20C |
| Dye Reagent | 120 uL | -20C |
| ALT Enzyme | 120 uL | -20C |
| Cosubstrate | 600 uL | -20C |
| Alanine Standard | 400 uL | -20C |
