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Amplite® Fluorimetric Alanine Aminotransferase Assay Kit

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).
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Catalog Number13802
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Component A: ALT Enzyme Mixture1 bottle (lyophilized powder)
Component B: ALT Assay Bufferbottle (10 mL)
Component C: NAD1 vial
Component D: ALT Positive Control1 vial (10 U)

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.

BL BL TS TS
ALT1 ALT1 ... ...
ALT2 ALT2 ... ...
ALT3 ALT3    
ALT4 ALT4    
ALT5 ALT5    
ALT6 ALT6    
ALT7 ALT7    

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

Well Volume Reagent
ALT1 - ALT7 50 µL Serial Dilution (1 to 1000 mU/mL)
BL 50 µL DPBS with 0.1% BSA
TS 50 µL Test 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 1 citations: Citation Explorer
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
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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
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Journal: Clin Gastroenterol Hepatol. (2012)
Transient elastographic evaluation in adult subjects without overt liver disease: influence of alanine aminotransferase levels
Authors: Kumar M, Sharma P, Garg H, Kumar R, Bhatia V, Sarin SK.
Journal: J Gastroenterol Hepatol (2011): 1318
The cross-sectional association between insulin resistance and circulating complement C3 is partly explained by plasma alanine aminotransferase, independent of central obesity and general inflammation (the CODAM study)
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Journal: Eur J Clin Invest (2011): 372
Nonalcoholic fatty liver disease is associated with blood pressure in hypertensive and nonhypertensive individuals from the general population with normal levels of alanine aminotransferase
Authors: Lopez-Suarez A, Guerrero JM, Elvira-Gonzalez J, Beltran-Robles M, Canas-Hormigo F, Bascunana-Quirell A.
Journal: Eur J Gastroenterol Hepatol (2011): 1011
Ferritin/alanine aminotransferase ratio as a possible marker for predicting the prognosis of acute liver injury
Authors: Ozawa E, Abiru S, Nagaoka S, Yano K, Komori A, Migita K, Yatsuhashi H, Taura N, Ichikawa T, Ishibashi H, Nakao K.
Journal: J Gastroenterol Hepatol (2011): 1326
Changes in serum levels of HBV DNA and alanine aminotransferase determine risk for hepatocellular carcinoma
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Journal: Gastroenterology (2011): 1240
Association of serum alanine aminotransferase and gamma-glutamyltransferase levels within the reference range with metabolic syndrome and nonalcoholic fatty liver disease
Authors: Oh HJ, Kim TH, Sohn YW, Kim YS, Oh YR, Cho EY, Shim SY, Shin SR, Han AL, Yoon SJ, Kim HC.
Journal: Korean J Hepatol (2011): 27
Non-alcoholic fatty liver disease's prevalence and impact on alanine aminotransferase associated with metabolic syndrome in the Chinese
Authors: Hou XH, Zhu YX, Lu HJ, Chen HF, Li Q, Jiang S, Xiang KS, Jia WP.
Journal: J Gastroenterol Hepatol (2011): 722