Amplite® Colorimetric Aspartate Aminotransferase (AST) Assay Kit

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Overview SDS Protocol

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® Colorimetric 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 blue color product through an enzyme coupled reaction cycle. The signal can be read by an absorbance microplate reader at an absorbance ratio of A570 nm to A610 nm. With the Amplite® Colorimetric 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

Absorbance microplate reader

Absorbance	570/610 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare AST working solution (50 µL)
Add AST standards or test samples (50 µL)
Incubate at 37ºC for 30 - 120 minutes
Monitor absorbance increase at the absrobance ratio of A_570nm /A_610nm

Important notes
Thaw the kit components 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 into the vial of 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/13801

Add 5 μL of 100 U/mL AST standard solution into 997 μL DPBS buffer with 0.1% BSA to generate 500 mU/mL AST standard solution (AST7). Take 500 mU/mL AST standard solution (AST7) and perform 1:2 serial dilutions in DPBS buffer with 0.1% BSA to get serial diluted AST standards (AST6 - AST1).

PREPARATION OF WORKING SOLUTION

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

2. Add 10 mL of AST Assay Buffer (Component B) into the bottle of AST Enzyme Mixture (Component A), and mix well. Then, add the whole vial of 100X NAD solution into the AST Enzyme Mixture solution to make 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. Avoid exposure to light. Note: Alternatively, one can make a 50X of AST Enzyme Mixture stock solution by adding 200 μL of H2O into the bottle of AST Enzyme Mixture (Component A), and then prepare the AST working solution by mix 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 clear bottom 96-well microplate. AST= AST Standards (AST1 - AST7, 7.8 to 500 mU/mL), BL=Blank Control, TS=Test Samples.

BL	BL	TS	TS
AST1	AST1	...	...
AST2	AST2	...	...
AST3	AST3
AST4	AST4
AST5	AST5
AST6	AST6
AST7	AST7

Table 2. Reagent composition for each well. Note: The AST standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.

Well	Volume	Reagent
AST1 - AST7	50 µL	Serial Dilutions (7.8 to 500 mU/mL)
BL	50 µL	DPBS with 0.1% BSA
TS	50 µL	test sample

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.
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.
Incubate the reaction at 37ºC for 30 - 120 minutes, protected from light. Note: The background of Blank Control increases with time and temperature.
Monitor the absorbance increase with an absorbance plate reader at the absorbance ratio of A_570nm /A_610nm.

Images

Figure 1. AST dose response was measured with Amplite® Colorimetric Aspartate Aminotransferase (AST) Assay Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices).

Citations

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Journal: Bioscience Reports (2020)

Spermidine confers liver protection by enhancing NRF 2 signaling through a MAP 1S-mediated non-canonical mechanism
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Journal: Hepatology (2019)

A mouse model study of toxicity and biodistribution of a replication defective adenovirus serotype 5 virus with its genome engineered to contain a decoy hyper binding site to sequester and suppress oncogenic HMGA1 as a new cancer treatment therapy
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Journal: PloS one (2018): e0192882

In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering
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