L-Glutamate Assay Kit, BioAssay™

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 = 66|2. Reagents supplied are ready to use.|3. Measurement range = 10 - 1,500mg/L|4. 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.||Reactions:|| (L-Glutamate oxidase)|1. L-Glutamate + H2O + O2 --------------------------------> a-Ketoglutarate + NH3 + H2O2 || (Peroxidase)|2. H2O2 + TOOS + 4-AA ------------------> Purple end-product|||Kit Components:|368991A: Enzyme Reagent, R1, 1 x 30ml|368991B: Enzyme Reagent, R2 1 x 30ml|368991C: 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.||6. Measure the absorbance of each tube at 555nm