This product includes 1 ml of 10 x dye C53. It is for 1000 assays using 384-well plates or 250 assays using 96-well plates. Cuvettes may also be used for the assays.
The Amino Acid Assay Kit is for measurement of micromolar to millimolar concentrations of amino acids. The assay sensitivity is 0.01 mM for His and Cys and less than 0.5 mM for most amino acids. The assay is based on the principle that amino acids interacts with dye C53 and enhance the fluorescence intensity at 535 nm (excitation at 485 nm).
The assay is compatible with HEPES buffer, low concentrations of non-ionic detergent (<0.01%), MgCl2(< 5 mM), CaCl2 (<5 mM) and phosphate (< 1 mM). The assay is not compatible with Tris buffer or other primary amine buffers. It is not compatible with DTT or EDTA.
The assay kit (catalog number AAK1000) includes 1 ml of 10 x dye C53. It is for 1000 assays using 384- well plates or 250 assays using 96-well plates. Cuvettes may also be used for the assays.
MEASUREMENT OF AMINO ACIDS USING 96-WELL PLATES
Amino Acid Standard Curve
1. Prepare standard amino acid solutions with a series of concentrations from 0.01 to 10 mM in 10 mM HEPES, pH7.5.
2. Prepare 1 x dye C53 dye by dilution of the 10 x dye C53 with water (10-fold dilution).
3. Mix 160 µl of the amino acid solution with 40 µl of 1 x dye C53 in the wells of a 96-well plate for 5 min and read the fluorescence at 535 nm (excitation at 485 nm).
Data Analysis
Plot the fluorescence intensity Fc and the amino acid concentration [Amino acid] to generate the linear standard curve.
Fc = a [Amino acid] + b
Where the Fc values are from experimental data, the a and b values are from the linear fitting between the Fc values and the amino acid concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the acid concentrations in the unknown samples using the Fc values from the unknown samples and the a and b values from the standard curve.