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Amplite® Colorimetric L-Alanine Assay Kit

L-alanine dose response was measured at 30-minute intervals using Amplite® Colorimetric L-Alanine Assay Kit on a white clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
L-alanine dose response was measured at 30-minute intervals using Amplite® Colorimetric L-Alanine Assay Kit on a white clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
L-alanine dose response was measured at 30-minute intervals using Amplite® Colorimetric L-Alanine Assay Kit on a white clear 96-well plate using a SpectraMax microplate reader (Molecular Devices).
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


L-alanine (L-Ala) plays a crucial role as a building block of important proteins. L-alanine is mostly synthesized by the muscle cells from lactic acid and absorbed into blood via the liver. It is converted into pyruvate by glutamic-pyruvic transaminase to enter the metabolic mainstream. L-Ala is critical for the production of glucose and hence blood sugar management, and plays an important role on the immune system and prevention of kidney stones. Insufficiency of L-alanine is usually a sign of poor nutrition, low protein diet, as well as stress. AAT Bioquest's Amplite® Colorimetric L-Alanine Assay Kit offers a sensitive colorimetric assay for quantifying L-alanine in biological samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which can be detected by Quest Fluor™ L-Alanine Sensor in an absorbance microplate reader at 575 nm.

Platform


Absorbance microplate reader

Absorbance575 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare L-Alanine standards or test samples (50 µL)
  2. Add L-Alanine working solution (50 µL)
  3. Incubate at 37°C for 1 hour to 2 hours

  4. Monitor absorbance intensity at 575 nm
Important Note

To achieve the best result, it is strongly recommended to use the white clear plates. Thaw kit components at room temperature before use.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Quest Fluor™ L-Alanine Sensor stock solution (200X)

Add 50 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.

L-Alanine standard solution

Add 10 µL of 100 mM L-Alanine Standard (Component D) into 490 µL of PBS (pH 7.0) to get 2 mM L-Alanine standard solution. Add 7.5 µL of 100 mM L-Alanine Standard (Component D) into 492.5 µL of PBS (pH 7.0) to get 1.5 mM L-Alanine standard solution.

PREPARATION OF WORKING SOLUTION

L- Alanine standard serial dilutions

Use 2 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (2000, 1000, 500, 250, 125 uM). Use 1.5 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (1500, 750 uM).

L-Alanine working solution
  1. Add 5 mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well.

  2. Add 100 μL of ddH2O into one Enzyme Mix2 vial (Component B2) and mix well.

  3. Transfer entire vial (100 μL) of Enzyme Mix2 and 25 uL of 200X L-Alanine Sensor stock solution into the Enzyme Mix1 bottle and mix well to make L-Alanine working solution. Note: L-Alanine working solution is not stable - use it promptly and avoid direct exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of L-Alanine standards and test samples in a white clear 96-well microplate. AS= L-Alanine Standard (AS1 - AS7, 125 to 2000 µM), BL=Blank Control, TS=Test Sample.

BLBLTSTS
AS1AS1......
AS2AS2......
AS3AS3
AS4AS4
AS5AS5
AS6AS6
AS7AS7

Table 2. Reagent composition for each well.

WellVolumeReagent
AS1 - AS750 µL

Serial Dilutions (125 to 2000 µM)

BL50 µLPBS
TS50 µLtest sample
  1. Prepare L-Alanine standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
  2. Add 50 µL of L-Alanine working solution to each well of L-Alanine standard, blank control, and test samples to make the total L-Alanine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Alanine working solution into each well instead, for a total volume of 50 µL/well. Note: Run the L-Alanine assay at pH 6.5 to 7.0.
  3. Incubate the reaction at 37°C for 1 hour to 2 hours.

  4. Monitor the absorbance increase with an absorbance plate reader at 575 nm.

Images


Citations


View all 2 citations: Citation Explorer
Colorectal Cancer Progression Is Potently Reduced by a Glucose-Free, High-Protein Diet: Comparison to Anti-EGFR Therapy. Cancers 2021, 13, 5817
Authors: Skibbe, K and Brethack, AK and S{\"u}nderhauf, A and Ragab, M and Raschdorf, A and Hicken, M and Schlichting, H and Preira, J and Brandt, J and Castven, D and others,
Journal: (2021)
Causes and consequences of a glutamine induced normoxic HIF1 activity for the tumor metabolism
Authors: Kappler, Matthias and Pabst, Ulrike and Weinholdt, Claus and Taubert, Helge and Rot, Swetlana and Kaune, Tom and Kotrba, Johanna and Porsch, Martin and G{\"u}ttler, Antje and Bache, Matthias and others,
Journal: International journal of molecular sciences (2019): 4742

References


View all 39 references: Citation Explorer
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