Enzymatic Method for Determining Alanine (Alanine Assay)

This assay protocol is suitable for the colorimetric/fluorometric detection of Alanine in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Alanine Assay Kit (MAK001). Alanine 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 alanine present. Typical detection ranges for this kit are 2-10 nmole (colorimetric) and 0.2-1 nmole (fluorometric).

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

Alanine Assay Buffer 25 mL
Catalog Number MAK001A

Alanine Probe, in DMSO 0.2 mL
Catalog Number MAK001B

Alanine Converting Enzyme 1 vl
Catalog Number MAK001C

Alanine Development Mix 1 vl
Catalog Number MAK001D

Alanine Standard, 10 µmole 1 vl
Catalog Number MAK001E

Reagents and Equipment Required but Not Provided:

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

Fluorescence or spectrophotometric multiwell plate reader

10 kDa Molecular Weight Cut-Off (MWCO) Spin Filter (Z706345 or equivalent)

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Alanine Assay Buffer – Allow buffer to come to room temperature before use.

Alanine Probe – Warm to room temperature to melt frozen solution prior to use. Store protected from light and moisture at –20 °C. Upon thawing, the Alanine Probe is ready-to-use in the colorimetric assay.

For the fluorescence assay, dilute an aliquot of the colorimetric Alanine Probe Solution 5 to 10-fold with Alanine Assay Buffer, just prior to use. This will reduce the background of the fluorescence assay.

Alanine Converting Enzyme – Reconstitute in 220 µL of Alanine Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Keep cold while in use and protect from light. Use within 2 months of reconstitution.

Alanine Development Mix – Reconstitute in 220 µL of Alanine Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Use within 2 months of reconstitution.

Alanine Standard – Reconstitute in 100 µL of water to generate a 100 mM (100 nmole/µL) Alanine Standard Solution. Mix well by pipetting, then aliquot and store at –20 °C. 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.

Alanine Standards for Colorimetric Detection
Dilute 10 µL of the 100 mM (100 nmole/µL) Alanine Standard Solution with 990 µL of water to prepare a 1 mM (1 nmole/µL) standard solution. Add 0, 2, 4, 6, 8, 10 µL of the 1 mM alanine standard solution into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Alanine Assay Buffer to each well to bring the volume to 50 µL.

Alanine Standards Fluorometric Detection Prepare a 1 mM standard solution as for the colorimetric assay. Take 100 µL of the 1 mM alanine standard solution and add to 900 µL of water to make a 0.1 mM alanine standard solution. Add 0, 2, 4, 6, 8, 10 µL of the 0.1 mM alanine standard solution into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add Alanine Assay Buffer to each well to bring the volume to 50 µL.

Sample Preparation
Both the colorimetric and fluorometric assays require 50 µL of sample for each reaction (well).

Serum samples should be deproteinized before use in assay with a 10 kDa MWCO spin filter. 10–50 µL of deproteinized serum samples can be directly diluted to a final volume of 50 µL with the Alanine Assay Buffer.

Tissue or cells (1 x 10⁶) can be homogenized in 100 µL of the Alanine Assay Buffer. Centrifuge the samples at 13,000 x g for 10 minutes to remove insoluble material. Bring samples to a final volume of 50 µL with Alanine Assay Buffer.
Notes: Samples other than serum may also be deproteinized with a 10 kDa MWCO spin filter prior to addition to the reaction.

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

Include a blank sample for each sample by omitting the Alanine Converting Enzyme in the Reaction Mix.

Assay Reaction

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

Table 1

Reagent	Blank Sample	Samples and Standards
Alanine Assay Buffer	46 µL	44 µL
Alanine Converting Enzyme	-	2 µL
Alanine Development Mix	2 µL	2 µL
Alanine Probe	2 µL	2 µL

2. Add 50 µL of the appropriate Reaction Mix to each of the blank, standard, and test wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 60 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) alanine standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate alanine standards to plot a standard curve.
Note: A new standard curve must be set up each time the assay is run.

Subtract the blank sample value from the sample reading to obtain the corrected measurement. Using the corrected measurement, the amount of alanine present in the sample may be determined from the standard curve.

Concentration of Alanine

S_a/S_v = C

S_a = Amount of alanine in unknown sample (nmole) from standard curve
S_v = Sample volume (µL) added into the wells
C = Concentration of alanine in sample

L-Alanine molecular weight: 89.09 g/mole.

Sample Calculation
Amount of alanine (S_a) = 5.84 nmole
Sample volume (S_v) = 50 µL
Concentration of alanine in sample

5.84 nmole/50 µL = 0.1168 nmole/µL
0.1168 nmole/µL x 89.09 ng/nmole= 10.4 ng/µL

Figure 1. Colorometric Standard Curve

Figure 2. Fluorometric Standard Curve