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Enzymatic Method for Determining L-Amino Acids (L-Amino Acid Quantitation Assay)

This assay protocol is suitable for the colorimetric/fluorometric detection of L-amino acids in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Amino Acid Quantitation Kit (MAK002). L-amino acid concentration is determined by a coupled enzyme assay, which results in a colorimetric (570 nm)/fluorometric (λex = 535/λem = 587 nm) product, proportional to the L-amino acids present. Typical detection ranges for this kit are 8–40 nmole (colorimetric) and 0.8–4.0 nmole (fluorometric). This assay is not suitable for the detection of glycine.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents

L-Amino Acid Assay Buffer                  25 mL
   Catalog Number MAK002A

L-Amino Acid Probe, in DMSO             0.2 mL
   Catalog Number MAK002B

L-Amino Acid Enzyme Mix                       1 vl
   Catalog Number MAK002D

L-Amino Acid Standard, 4 nmole/µL      0.3 ml
   Catalog Number MAK002E

Reagents and Equipment Required but Not Provided:

96 well flat-bottom plate – It is recommended to use black plates with clear bottoms (Catalog Number M5686 or equivalent) for fluorescence assays and clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.

Fluorescence or spectrophotometric multiwell plate reader

Preparation Instructions

Briefly centrifuge vials before opening. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

L-Amino Acid Assay Buffer – Allow buffer to come to room temperature before use.

L-Amino Acid Probe – Thaw at room temperature to melt solution prior to use. Aliquot and store protected from light and moisture at –20 °C.

L-Amino Acid Enzyme Mix – Reconstitute with 220 µL of L-Amino Acid Assay Buffer. Mix well by pipetting, then aliquot and store, protected from light, at –20 °C. Use within 2 months of reconstitution and keep cold while in use.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

All samples and standards should be run in duplicate.

L-Amino Acid Standards for Colorimetric Detection
Add 0, 2, 4, 6, 8, and 10 µL of the L-Amino Acid Standard (4 nmole/µL) into a 96 well plate, generating 0 (blank), 8, 16, 24, 32, and 40 nmole/well standards. Add L-Amino Acid Assay Buffer to each well to bring the volume to 50 µL.
Note: The standard is an equimolar mixture of all proteinogenic amino acids with the exception of glycine.

L-Amino Acid Standards for Fluorometric Detection
Dilute 20 µL of the L-Amino Acid Standard (4 nmole/µL) with 180 µL of L-Amino Acid Assay Buffer to prepare a 0.4 nmole/µL standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the L-Amino Acid standard solution into a 96 well plate, generating 0 (blank), 0.8, 1.6, 2.4, 3.2, and 4.0 nmole/well standards. Add L -Amino Acid Assay Buffer to each well to bring the volume to 50 µL.
Note: The standard is an equimolar mixture of all proteinogenic amino acids with the exception of glycine.

Sample Preparation
Tissue (10 mg) or cells (2 x 106) should be rapidly homogenized in 4 volumes of cold L-Amino Acid Assay buffer. Centrifuge at 13,000 x g for 10 minutes at 4 °C to remove insoluble material.

Serum and other liquid samples can be directly added to the wells.

Bring samples to a final volume of 50 µL with L-Amino Acid Assay Buffer.

For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.

Assay Reaction


1. Set up the Master Reaction Mix according to the scheme in Table 1. 50 µL of the Master Reaction Mix is required for each reaction (well).

Table 1.

Reagent Volume
L-Amino Acid Assay Buffer 46 µL
L-Amino Acid Probe 2 µL
L-Amino Acid Enzyme Mix 2 µL



2. Add 50 µL of the Master Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at 37 °C. Protect the plate from light during the incubation.

3. For colorimetric assays, measure the absorbance at 570 nm (A570). For fluorometric assays, measure fluorescence intensity (λex = 535/λem = 587 nm).

Results

Calculations
The background for the assays is the value obtained for the 0 (blank) L-Amino Acid Standard. Correct for the background by subtracting the 0 (blank) value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate L-Amino Acid standards to plot a standard curve.
Note: A new standard curve must be set up each time the assay is run.

Concentration of L -Amino Acids

    Sa/Sv = C

Sa = Amount of L-Amino Acids in unknown sample (nmole) from standard curve
Sv = Sample volume (µL) added into the wells
C = Concentration of L-Amino Acids in sample

Sample Calculation
Amount of L-Amino Acid (Sa) = 5.84 nmole (from standard curve)
Sample volume (Sv) = 50 µL
Concentration of L-Amino Acids in sample

5.84 nmole/50 µL = 0.1168 nmole/µL

Materials

     
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