The L-Glutamate Assay Kit is a colorimetric assay for the determination L-Glutamic Acid concentration in food samples.
Features of the Kit:1. Number of tests = 662. Reagents supplied are ready to use
3. Measurement range = 10 - 1,500mg/L4. No sample pre-treatment required for samples containing up to 1g/L ascorbic acid
Principle of the Assay:R1 enzyme reagent is added to the sample to remove ascorbic acid/Vitamin C from samples via the action of ascorbic acid oxidase. Addition of the R2 enzyme reagent converts L-glutamate in the sample to alpha-ketoglutarate, ammonia and hydrogen peroxide (reaction 1) via the action of L-glutamic acid oxidase. In the presence of peroxidase, the reaction of hydrogen peroxide with TOOS and 4-aminoantipyrine produces a purple colored dye product (reaction 2) whose absorbance is measured measured spectrophotometrically at 555nm. The intensity of the color obtained is directly proportional to L-glutamic acid concentration.
(Peroxidase)2. H2O2 + TOOS + 4-AA ------------------> Purple end-product
Kit Components:368991A: Enzyme Reagent, R1, 1 x 30ml368991B: Enzyme Reagent, R2 1 x 30ml368991C: L-Glutamic Acid Standard Solution (250mg/L), 1x500ul
Storage and Stability:Store all kit components in the dark at 4°C. Shelf life is 6 months from time of receipt
Sample Preparation:1. Dilute liquid foods with purified water to adjust the L-Glutamic Acid concentration to 10 -1,500mg/L (e.g., 100-200-fold dilution for soy sauce)
2. Cut solid foods, such as cheese and sausage, into pieces. Mix these pieces with 10-20 times the amount of purified water or phosphate buffer. Cool and filter the mixture. Dilute the filtrates 2-5 fold with purified water for sample preparation. For turbid samples, repeat filtration and centrifugation. Protein removal is not needed
3. Media can be measured without dilution. Samples with concentrations above the measurement range need to be diluted with purified water.
Assay Procedure:1. Add 10ul each of sample, standard solution and purified water to tubes.
2. Add 450ul of R1 enzyme reagent to the tubes; mix tube contents.
3. Allow the tubes to stand at 20-30°C for 20 minutes
Note: For samples for which ascorbic acid does not need to be removed, skip step 3 and proceed to step 4.
4. Add 450ul of R2 enzyme reagent to each tube; mix tube contents
Note: Since the color of the samples (especially dark samples) may affect absorbance, prepare also a tube containing 10ul of sample and 900ul of purified water (label this as sample dye solution).
5. Allow the tubes to stand at 20-30°C for 20 minutes.
