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Amplite® Fluorimetric Asparaginase Activity Assay Kit

The Amplite® Fluorimetric Asparaginase Activity Assay Kit provides a simple and straightforward procedure for measuring Asparaginase activity in a variety of biological samples. Asparaginase activity is determined by a coupled enzyme assay, which results in the formation of a fluorogenic product with Ex/Em=540/590nm. The amount of the fluorogenic product formed is proportional to the aspartate generated by asparaginase enzyme. 1 unit (U) is the amount of enzyme that catalyzes the reaction of 1 µmol of substrate per minute. Asparaginase is an essential enzyme that catalyzes the hydrolysis of the non-essential amino acid asparagine to aspartate and ammonia. It plays a crucial role in cellular functions, particularly in hematopoietic cells which rely on exogenous asparagine for protein synthesis. Asparaginase is found in plants, microorganisms, and certain animals, but does not occur naturally in humans, making it a valuable therapeutic agent in medicine and an essential tool in various industries. Asparaginase is used to treat acute lymphocytic leukemia (ALL) by starving tumor cells of needed nutrients and slowing tumor cell growth. Depletion of circulating asparagine by asparaginase induces cell cycle arrest and apoptosis in malignant cells, offering a targeted approach to cancer treatment. Beyond its medical applications, asparaginase is also used in the food industry to reduce the formation of acrylamide, a potentially carcinogenic compound, in starchy and fried foods. The versatility of this enzyme extends to various research fields, including biotechnology and biochemistry.

Example protocol

AT A GLANCE

Important Note

Thaw all the kit components at room temperature before starting the experiment. 

Protocol Summary
  1. Prepare test samples and serially dilute Aspartate or Asparaginase standards (50 μL).

  2. Add the Asparaginase working solution (50 μL).

  3. Incubate at 37 °C for 10-30 minutes.

  4. Monitor the fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm).

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

Amplite® Red Substrate Stock Solution (100X)
  1. To prepare a 100X Amplite® Red Substrate stock solution, add 60 µL of DMSO (Component G) to the vial of Amplite® Red Substrate (Component A).
Enzyme Mix 2 Stock Solution (50X)
  1. To prepare a 50X Enzyme Mix 2 stock solution, add 100 µL of ddH2O to the Enzyme Mix 2 vial (Component B2).
Conversion Mix Stock Solution (100X)
  1. To prepare a 100X Conversion Mix stock solution, add 50 µL of ddH2O to the Conversion Mix vial (Component D).
Aspartate Standard Solution (10mM)
  1. To prepare at 10 mM aspartate standard solution, add 100 µL of ddH2O to the Aspartate Standard vial (Component E).
Asparaginase Positive Control Stock Solution
  1. Reconstitute Asparaginase Positive Control (Component F) with 100 µL of ddH2O, and mix well. Aliquot the solution and store it at -20 °C.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11799

Aspartate Standard
Add 12 µL of a 10 mM aspartate standard to 288 µL of 1X PBS buffer to prepare a 400 µM aspartate solution (STD7). Next, perform 1:2 serial dilutions in 1X PBS buffer to obtain the serially diluted aspartate standards (STD6-STD1).

PREPARATION OF WORKING SOLUTION

Asparaginase Working Solution
  1. Add 5 mL of Assay Buffer (Component C) to the Enzyme Mix 1 bottle (Component B1) and mix well.

  2. Add 100 μL of Enzyme Mix 2 stock solution to the same bottle and mix well.

  3. Add 50 μL of the 100X Conversion Mix stock solution to the same bottle and mix well.

  4. Add 50 µL of the Amplite® Red Substrate stock solution to the same bottle and mix well.

    Note: The working solution is not stable, use it promptly, and avoid direct exposure to light.

    Note: Alternatively, one can make a 50X Enzyme Mix 1 stock solution by adding 100 μL of ddH2O into the bottle of Enzyme Mix 1 (Component B1) and then prepare the Asparaginase working solution by mixing the Enzyme Mix 1 stock solution with other components listed above in the 'Asparaginase Working Solution' proportionally.

Asparaginase Positive Control
  1. To prepare one or more Asparaginase positive controls alongside the test sample, dilute the Asparaginase Positive Control stock solution using a dilution factor between 100 and 1000 times in 1X PBS. For example, for a 250-fold dilution, mix 4 µL of Asparaginase Positive Control stock solution with 996 µL of 1X PBS.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Aspartate standards and test samples in a 96-well clear bottom white microplate. (STD = Aspartate Standards (STD1-STD7, 6.25 to 400 µM), BL = Blank Control, TS = Test Samples)

BL
BL
Positive Control
TS
STD 1
STD 1
...
...
STD 2
STD 2
...
...
STD 3
STD 3
STD 4
STD 4
STD 5
STD 5
STD 6
STD 6
STD 7
STD 7

Table 2. Reagent composition for each well.

Well
Volume
Reagent
STD 1 - STD 7 
50 µL
Serial Dilutions (6.25 to 400 µM) in PBS
BL 
50 µL
PBS
Asparaginase Positive Control 
50 µL
Asparaginase Positive Control in PBS
TS 
50 µL
Test Sample
  1. Prepare the Aspartate standards (STD 1-STD 7), blank controls (BL), Asparaginase Positive Control, and test samples (TS) according to the layout provided in Tables 1 and 2. When using a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of Asparaginase Working Solution to each well of the blank control, Asparaginase Positive Control, and test samples. If using a 384-well plate, add 25 µL of Asparaginase Working Solution to each well instead.

  3. Incubate at 37 °C for 10-30 minutes, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 540 nm/ 590 nm (Cutoff = 570 nm).

References

View all 49 references: Citation Explorer
Monitoring asparaginase activity to improve the management of patients with acute lymphoblastic leukemia: Experience in one center.
Authors: García Morin, Marina and Melero Guardia, Paula and Bardón-Cancho, Eduardo J and Zapico Muñiz, Edgar and Cela, Elena
Journal: Anales de pediatria (2024): 65-66
Determination of l-Asparaginase Activity and Its Therapeutic Monitoring in Children with Hematological Malignancies in a Single Croatian Center.
Authors: Lenicek Krleza, Jasna and Katusic Bojanac, Ana and Jakovljevic, Gordana
Journal: Diagnostics (Basel, Switzerland) (2024)
Improved HPLC method with automated pre-column sample derivatisation for serum pegylated L-asparaginase activity measurement in paediatric acute lymphoblastic leukaemia patients.
Authors: Tan, Yan Qi and Loh, C-Khai and Mohd Saffian, Shamin and Makpol, Suzana
Journal: Journal of pharmaceutical and biomedical analysis (2024): 116243
Allergic Reactions to E. coli Asparaginase are Associated with Decreased Asparaginase Activity in an Indonesian Pediatric Population with ALL.
Authors: Sari, Nur Melani and Berbudi, Afiat and Susanah, Susi and Reniarti, Lelani and Supriyadi, Eddy and Kaspers, Gertjan J L and Buddington, Randal K and Howard, Scott and Idjradinata, Ponpon
Journal: Asian Pacific journal of cancer prevention : APJCP (2023): 2773-2780
Our Experiences with Asparaginase Activity Measurements in Children with Lymphoblastic Diseases.
Authors: Müller, Judit and Egyed, Petra and Erdelyi, Daniel and Kovacs, Krisztian and Mudra, Katalin and Szabo, Sandor and Egyed, Balint and Gabor, Kovacs
Journal: Children (Basel, Switzerland) (2023)
Page updated on June 9, 2025

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Catalog Number11799
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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

Aspartate dose response was measured using the Amplite® Fluorimetric Asparaginase Activity Assay Kit on a 96-well clear bottom black solid microplate using a fluorescence microplate reader (Ex/Em = 540/590, Cutoff = 570 nm).
Aspartate dose response was measured using the Amplite® Fluorimetric Asparaginase Activity Assay Kit on a 96-well clear bottom black solid microplate using a fluorescence microplate reader (Ex/Em = 540/590, Cutoff = 570 nm).
Aspartate dose response was measured using the Amplite® Fluorimetric Asparaginase Activity Assay Kit on a 96-well clear bottom black solid microplate using a fluorescence microplate reader (Ex/Em = 540/590, Cutoff = 570 nm).