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Amplite® Fluorimetric Alanine Aminotransferase Assay Kit

Alanine aminotransferase (ALT), also called serum glutamate pyruvic transaminase (GPT), is a member of transferase family. It catalyzes the reversible transfer of an alpha-amino group between alanine and glutamate, and is an important enzyme in amino acid metabolism. ALT is found mainly in liver and small amount in heart, muscle, and kidneys. In healthy subjects, serum ALT levels are low. However, when cells are damaged, such as acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction, ALT may leak into the blood stream and the ALT levels are significantly elevated. Therefore, determination of serum ALT level has great clinical and diagnostic significance. Amplite® Fluorimetric Alanine Aminotransferase Assay Kit provides a quick and sensitive method for the measurement of ALT in various biological samples. ALT catalyzes the reaction of alanine and α-ketoglutarate to pyruvate and glutamate. The product 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 Alanine Aminotransferase Assay Kit as little as 4 mU/mL ALT was detected in a 100 µL reaction volume. The assay is robust, and can be readily adapted for a wide variety of applications.

Example protocol

AT A GLANCE

Protocol summary

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

Important notes
Thaw one bottle 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. ALT standard solution:
Add 100 µL DPBS into the vial of ALT Positive Control (Component D) to make 100 U/mL ALT stock solution.

PREPARATION OF STANDARD SOLUTION

ALT standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13802

Add 10 µL of 100 U/mL ALT standard solution into 990 µL DPBS buffer with 0.1% BSA to generate 1000 mU/mL ALT standard solution (ALT7). Take the 1000 mU/mL ALT standard solution and perform 1:3 serial dilutions to get serial dilutions of ALT standard (ALT6 - ALT1).

PREPARATION OF WORKING SOLUTION

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

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

3. ALT working solution:
Add the whole vial of 100X NAD solution (from 1) into the ALT Enzyme Mixture solution (from 2) to have ALT working solution.

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

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of ALT standards and test samples in a solid black 96-well microplate. ALT = ALT standard (ALT1 - ALT7, 1 to 1000 mU/mL); BL = blank control; TS = test sample.

BLBLTSTS
ALT1ALT1......
ALT2ALT2......
ALT3ALT3  
ALT4ALT4  
ALT5ALT5  
ALT6ALT6  
ALT7ALT7  

Table 2. Layout of ALT standards and test samples in a solid black 96-well microplate.

WellVolumeReagent
ALT1 - ALT750 µLSerial Dilution (1 to 1000 mU/mL)
BL50 µLDPBS with 0.1% BSA
TS50 µLTest Sample
  1. Prepare ALT standards (ALT), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according the the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Dilute the test samples to the appropriate concentration range (indicated by the ALT standards) in DPBS buffer with 0.1% BSA if needed.

  2. Add 50 µL of ALT working solution into each well of ALT standard, blank control, and test samples to make the total ALT assay volume of 100 µL/well. For a 384-well microplate, add 25 µL of ALT working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at 37°C for 30 min to 60 minutes, protected from light. Note: The background of Blank Control will increase with time.

  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).

Citations

View all 2 citations: Citation Explorer
Reparative Efficacy of Liposome-Encapsulated Oleanolic Acid against Liver Inflammation Induced by Fine Ambient Particulate Matter and Alcohol in Mice
Authors: Wei, Ching-Ting and Wang, Yu-Wen and Wu, Yu-Chiuan and Lin, Li-Wei and Chen, Chia-Chi and Chen, Chun-Yin and Kuo, Shyh-Ming
Journal: Pharmaceutics (2022): 1108
The receptor for advanced glycation endproducts (RAGE) contributes to severe inflammatory liver injury in mice
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

References

View all 226 references: Citation Explorer
Pegylated interferon alpha-2b plus ribavirin for Japanese chronic hepatitis C patients with normal alanine aminotransferase
Authors: Kainuma M, Furusyo N, Azuma K, Kajiwara E, Takahashi K, Nomura H, Tanabe Y, Satoh T, Maruyama T, Nakamuta M, Kotoh K, Shimoda S, Hayashi J.
Journal: Hepatol Res (2012): 33
Coexistence of non-alcoholic fatty liver disease with elevated alanine aminotransferase is associated with insulin resistance in young Han males
Authors: Wang R, Lu Q, Feng J, Yin F, Qin C, Liu B, Liu Y, Liu X.
Journal: Endocrine (2012): 70
Levels of Alanine Aminotransferase Confound Use of Transient Elastography to Diagnose Fibrosis in Patients with Chronic HCV Infection
Authors: Tapper EB, Cohen EB, Patel K, Bacon B, Gordon S, Lawitz E, Nelson D, Nasser IA, Challies T, Afdhal N.
Journal: Clin Gastroenterol Hepatol. (2012)
Gender differences in healthy ranges for serum alanine aminotransferase levels in adolescence
Authors: Poustchi H, George J, Esmaili S, Esna-Ashari F, Ardalan G, Sepanlou SG, Alavian SM.
Journal: PLoS One (2011): e21178
Updated thresholds for alanine aminotransferase do not exclude significant histological disease in chronic hepatitis C
Authors: Sanai FM, Helmy A, Dale C, Al-Ashgar H, Abdo AA, Katada K, AlMana H, Saadeh M, Al-Hussaini H, AlQuaiz M, Hashem A, AlSwat K, Bzeizi KI, Marotta PJ.
Journal: Liver Int (2011): 1039
Page updated on November 5, 2024

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

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