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Protokoll
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1. Standard Curve Preparations: For colorimetric assay, add 0, 2, 4, 6, 8, 10 μ l L-Amino Acid Standard into each well individually of a 96-well plate to generate 0, 8, 16, 24, 32, 40 nmol/well of L-Amino Acid Standard. Adjust volume to 50 μ l/well with L-Amino Acid Assay Buffer. For fluorometric assay, dilute the L-Amino Acid to 0.4 nmol/ μ l by adding 10 μ l of the L-Amino Acid to 90 μ l of L-Amino Acid Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 μ l into each well individually to generate 0, 0.8, 1.6, 2.4, 3.2, 4.0 nmol/well of the L-Amino Acid Standard. Adjust volume to 50 μ l/well with L-Amino Acid Assay Buffer. 2. Sample Preparations: Prepare test samples in 50 μ l/well with L-Amino Acid Assay Buffer in the 96-well plate. We suggest using several doses of your sample to ensure the readings are within the standard curve range. 3. Reaction Mix Preparation: Mix enough reagents for the number of assays performed: For each well, prepare a total 50 μ l Reaction Mix containing: 46 μ l L-Amino Acid Assay Buffer 2 μ l L-Amino Acid Probe 2 μ l L-Amino Acid Enzyme Mix, Mix well. 3. Add 50 μ l of the Reaction Mix to each well containing the L-Amino Acid standard or test samples. 4. Incubate the reaction for 30 minutes at 37 o C, protect from light. 5. Measure O.D. 570nm for colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a micro-plate reader. 6. Correct background by subtracting the value of the 0 L-amino acid control from all samples (The background reading can be significant and must be subtracted from sample readings). Then apply the sample readings to the L- amino acid standard curve to obtain the total amino acid amount. L-Amino Acid Concentration = A/Sv (nmol/ μ l or mM) Where A: L-Aamino acid amount (nmol) from the standard curve based on Absorbance OD570 or fluorescence of your samples. Sv: Sample volume ( μ l) you added into the sample wells. L-Amino Acid Standard Curve. Different doses of L-Amino Acids were measured according to the kit procedure. V. Related Products: Cholesterol/Cholesteryl Esters Quantitation Kit HDL, LDL/VLDL cholesterol assay Kit Glucose Assay Kit Lactate Assay Kit Glutathione Assay Kit NAD/NADH Assay Kit LDH-cytotoxicity Assay Kit Uric Acid Assay Kit Apoptosis Detection Kits Components K639-100 Cap Code Parts Number L-Amino Acid Assay Buffer L-Amino Assay Probe DMSO (Dried) L-Amino Acid Enzyme Mix L-Amino Acid Standard (4nmol/ μ l)* 25 ml 1 Vial 500 μ l 1 Vial 300 μ l WM Red Brown Green Yellow K639-100-1 K639-100-2 K639-100-3 K639-100-4 K639-100-5 0 0.6 1.2 1.8 0 8 16 24 32 40 Total L-Amino Acid (nmol/Well) O.D. 570 nm
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