Amplite™ Fluorimetric L-Alanine Assay Kit

Image Viewer
Analyze with Quest Graph™Plan a serial dilution
Close (X)
1e+4100010- Dose-responseData legend Generated with Quest Graph™ L-Alanine (uM) RFU Hover mouse to interact
L-alanine dose response was measured with Amplite™ Fluorimetric L-Alanine Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Loading...
 
Unit Size: Cat No: Price (USD): Qty:
200 Tests 13825 $295


Export item/cart as Excel file

Send item/cart as email
EXPORT TO EXCEL X

Export:
EXPORT TO EMAIL X
Important: We request your email address to ensure that the recipient(s) knows you intended for them to see the email, and that it is not junk mail.
Export:
Your Name*:
Your Email*:
Recipient Email*:
Your Personal Message:
Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
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 in 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™ Fluorimetric L-Alanine Assay Kit offers a sensitive fluorescent assay for quantifying L-alanine in biological samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which is detected by Quest Fluor™ L-Alanine Sensor with a fluorescence microplate reader at Ex/Em = 540/590 nm.




Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare test samples along with serially diluted L-Alanine standards (50 µL)
  2. Add equal volume of L-Alanine working solution (50 µL)
  3. Incubate at 37°C for 30 minutes to 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
To achieve the best result, it’s strongly recommended to use the black plates. Thaw kit components at room temperature before use.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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.

1. Quest Fluor™ L-Alanine Sensor stock solution (200X):
Add 55 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.

2. L-Alanine standard solution (1 mM):
Add 10 µL of 100 mM L-alanine (Component D) into 990 µL PBS (pH 7.0) to get 1 mM L-alanine solution.

Preparation of standard solution
L-Alanine standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13825

Add 100 µL of 1 mM L-Alanine standard solution into 900 µL PBS to make 100 µM L-alanine solution (AS7). Perform 1:2 serial dilutions to get serially diluted L-alanine standards (AS6 - AS1).

Preparation of working solution

1. Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well.

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

3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 uL of 200X L-alanine sensor stock solution into the Enzyme Mix 1 bottle; mix well. Note: The working solution is not stable. Use promptly and avoid direct exposure to light.

Sample experimental protocol

Table 1. Layout of L-alanine standards and test samples in a solid black 96-well microplate. AS = L-Alanine standard (AS1 - AS7, 1.5 to 100 µM); BL = blank control; TS = test sample.

BL BL TS TS
AS1 AS1 ... ...
AS2 AS2 ... ...
AS3 AS3    
AS4 AS4    
AS5 AS5    
AS6 AS6    
AS7 AS7    

Table 2. Reagent composition for each well.

Well Volume Reagent
AS1 - AS7 50 µL Serial Dilution (1.5 to 100 µM)
BL 50 µL 1X PBS Buffer
TS 50  Test 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 mixture at 37°C for 30 minutes to 1 hour.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off: 570 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate L-Alanine samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. L-alanine dose response was measured with Amplite™ Fluorimetric L-Alanine Assay Kit on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Reactivity of beta-methylamino-L-alanine in complex sample matrixes complicating detection and quantification by mass spectrometry
Authors: Glover WB, Liberto CM, McNeil WS, Banack SA, Shipley PR, Murch SJ.
Journal: Anal Chem (2012): 7946

Synthesis and characterization of silver/alanine nanocomposites for radiation detection in medical applications: the influence of particle size on the detection properties
Authors: Guidelli EJ, Ramos AP, Zaniquelli ME, Nicolucci P, Baffa O.
Journal: Nanoscale (2012): 2884

Detection of farnesyltransferase interface hot spots through computational alanine scanning mutagenesis
Authors: Perez MA, Sousa SF, Oliveira EF, Fernandes PA, Ramos MJ.
Journal: J Phys Chem B (2011): 15339

Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase
Authors: Tang Z, Liu P, Ma C, Yang X, Wang K, Tan W, Lv X.
Journal: Anal Chem (2011): 2505

Highly specific substrates of proteinase 3 containing 3-(2-benzoxazol-5-yl)-l-alanine and their application for detection of this enzyme in human serum
Authors: Wysocka M, Lesner A, Guzow K, Kulczycka J, Legowska A, Wiczk W, Rolka K.
Journal: Anal Chem (2010): 3883

In-capillary derivatization and stacking electrophoretic analysis of gamma-aminobutyric acid and alanine in tea samples to redeem the detection after dilution to decrease matrix interference
Authors: Su YS, Lin YP, Cheng FC, Jen JF.
Journal: J Agric Food Chem (2010): 120

SAFETY study: alanine aminotransferase cutoff values are set too high for reliable detection of pediatric chronic liver disease
Authors: Schwimmer JB, Dunn W, Norman GJ, Pardee PE, Middleton MS, Kerkar N, Sirlin CB.
Journal: Gastroenterology (2010): 1357

[Rapid detection of alanine aminotransferase with near-infrared spectroscopy]
Authors: Huang FR, Zhang J, Luo YH, Li SP, Zheng SF, Chen XD.
Journal: Guang Pu Xue Yu Guang Pu Fen Xi (2010): 2620

An electrochemical biosensor array for rapid detection of alanine aminotransferase and aspartate aminotransferase
Authors: Song MJ, Yun DH, Hong SI.
Journal: Biosci Biotechnol Biochem (2009): 474

Detection of the mitochondrial and catalytically active alanine aminotransferase in human tissues and plasma
Authors: Glinghammar B, Rafter I, Lindstrom AK, Hedberg JJ, Andersson HB, Lindblom P, Berg AL, Cotgreave I.
Journal: Int J Mol Med (2009): 621


View More Citations




Additional Documents

 
Safety Data Sheet (SDS)


Certificate of Analysis