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Amplite® Fluorimetric Aspartate Aminotransferase (AST) Assay Kit

AST dose response was measured with Amplite® Fluorimetric Aspartate Aminotransferase Assay Kit in a 96-well black plate using a Gemini fluorescence microplate reader (Molecular Devices).
AST dose response was measured with Amplite® Fluorimetric Aspartate Aminotransferase Assay Kit in a 96-well black plate using a Gemini fluorescence microplate reader (Molecular Devices).
AST dose response was measured with Amplite® Fluorimetric Aspartate Aminotransferase Assay Kit in a 96-well black plate using a Gemini fluorescence microplate reader (Molecular Devices).
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UNSPSC12352200

OverviewpdfSDSpdfProtocol


Aspartate aminotransferase (AST), also called serum glutamic oxaloacetic transaminase (GOT), is a member of transferase family. It catalyzes the reversible transfer of an alpha-amino group between aspartate and glutamate, and is an important enzyme in amino acid metabolism. AST is found in many body tissues such as liver, heart, muscle, kidneys, brain. In healthy subjects, serum AST levels are low. However, when cells are damaged, such as acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction, AST may leak into the blood stream and the AST levels are significantly elevated. Therefore, determination of serum AST level has great clinical and diagnostic significance. Amplite® Fluorimetric Aspartate Aminotransferase (AST) assay kit provides a quick and sensitive method for the measurement of AST in various biological samples. Aspartate transaminase catalyzes the reaction of aspartate and α-ketoglutarate to oxaloacetate and glutamate. The product L-glutamate is measured by the generation of a red fluorescent product through an enzyme coupled reaction cycle. The signal can be read by a fluorescence microplate reader. With the Amplite® Fluorimetric Aspartate Aminotransferase Assay Kit, we have detected as little as 2 mU/mL AST in a 100 µL reaction volume. The assay is robust, and can be readily adapted for a wide variety of applications.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare AST working solution (50 µL)
  2. Add AST standards or test samples (50 µL)
  3. Incubate at 37 °C for 20 - 30 min or RT for 60 min
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw one bottle each of Component A and B at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

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. AST standard solution (100 U/mL):
Add 100 µL DPBS Buffer to AST Positive Control (Component D) to make 100 U/mL AST standard solution.

PREPARATION OF STANDARD SOLUTION

AST standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13800

Add 3 µL of 100 U/mL AST standard solution into 997 µL DPBS buffer with 0.1% BSA to generate 300 mU/mL AST standard solution (AST7). Take 300 µL of 300 mU/mL AST standard solution to perform 1:3 serial dilutions to get serial dilutions of AST standard (AST6 - AST1). Note: The AST standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.

PREPARATION OF WORKING SOLUTION

1. NAD solution (100X):
Add 100 µL of ddH2O into the vial of NAD (Component C).

2. AST Enzyme Mixture solution:
Add 10 mL of AST Assay Buffer (Component B) into the bottle of AST Enzyme Mixture (Component A), and mix well.

3. AST working solution:
Add the whole vial of 100X NAD solution (from 1) into the AST Enzyme Mixture solution (from 2) to have AST working solution. Note: This AST working solution is enough for two 96-well plates. It is unstable at room temperature, and should be used promptly within 2 hours. Alternatively, one can make a 50X of AST Enzyme Mixture stock solution by adding 200 µL of H2O into the bottle of Component A, and then prepare the AST working solution by mixing the stock solution with assay buffer (Component B) and 100X NAD solution proportionally.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of AST standards and test samples in a solid black 96-well microplate. AST= AST Standards (AST1 - AST7, 0.3 to 300 mU/mL), BL=Blank Control, TS=Test Samples.

BLBLTSTS
AST1AST1......
AST2AST2......
AST3AST3  
AST4AST4  
AST5AST5  
AST6AST6  
AST7AST7  

Table 2. Reagent composition for each well.

WellVolumeReagent
AST1-AST750 µLSerial Dilution (0.3 to 300 mU/mL)
BL50 µLDPBS with 0.1% BSA
TS50 µLSample
  1. Prepare AST standards (AST), 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 AST working solution to each well of AST standard, blank control, and test samples to make the total AST assay volume of 100 µL/well. For a 384-well plate, add 25 µL of AST working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at 37°C for 20 - 30 minutes or room temperature for 60 minutes, protected from light. Note: The background of Blank Control increases with time and temperature.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).

 

Images


Citations


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Authors: Weinhage, Toni and Wirth, Timo and Sch{\"u}tz, Paula and Becker, Philipp and Lueken, Aloys and Skryabin, Boris V and Wittkowski, Helmut and Foell, Dirk
Journal: Frontiers in immunology (2020): 1157
Reparative effects of astaxanthin-hyaluronan nanoaggregates against retrorsine-CCl4-induced liver fibrosis and necrosis
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Journal: Molecules (2018): 726
In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering
Authors: Chang, Siou Han and Huang, Han Hsiang and Kang, Pei Leun and Wu, Yu Chian and Chang, Ming-Huang and Kuo, Shyh Ming
Journal: Acta Biomaterialia (2017)
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Authors: Tsai, Meng-Tsz and Katagiri, Naohiro and Ohbayashi, Norihiko and Iwasaki, Kenichi and Ohkohchi, Nobuhiro and Ding, Shih-Torng and Kanaho, Yasunori and Funakoshi, Yuji
Journal: Scientific reports (2017): 1--12

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