Amplite® Colorimetric Alanine Aminotransferase Assay Kit *Blue Color*
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
See also: Amplite® Reagents and Assay Kits, Transferases
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® Colorimetric 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 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 Alanine Aminotransferase Assay Kit as little as 10 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
Absorbance microplate reader
Absorbance | 570/610 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol Summary
- Prepare ALT working solution (50 µL)
- Add ALT standards or test samples (50 µL)
- Incubate at 37 °C for 60 mins to 120 mins
- Monitor absorbance increase at the absorbance ratio of A570nm/A610nm
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.
1. ALT standard solution (100 U/mL)
Add 100 µL DPBS into the vial of ALT Positive Control (Component D) to make 100 U/mL ALT standard solution.2. NAD solution (100X)
Add 100 µL of ddH2O into the vial of NAD (Component C) to have 100X NAD solution.PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13803
https://www.aatbio.com/tools/serial-dilution/13803
ALT standard
Add 10 µL of 100 U/mL ALT standard solution into 990 µL DPBS buffer with 0.1% BSA to generate 1 U/mL ALT standard solution (ALT7). Take 1 U/mL ALT standard solution and perform 1:2 serial dilutions in DPBS buffer with 0.1% BSA to get serial dilutions of ALT standard (ALT6 - ALT1).PREPARATION OF WORKING SOLUTION
- Add 10 mL of ALT Assay Buffer (Component B) into the bottle of ALT Enzyme Mixture (Component A) and mix well.
- Add the whole vial of 100X NAD solution into the ALT Enzyme Mixture solution to make 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 mix 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 clear, white, or black with clear bottom 96-well microplate. ALT= ALT Standards (ALT1 - ALT7, 15.6 to 1000 mU/mL), BL=Blank Control, TS=Test Samples
Table 2. Reagent composition for each well.
BL | BL | TS | TS |
ALT1 | ALT1 | ... | ... |
ALT2 | ALT2 | ... | ... |
ALT3 | ALT3 | ||
ALT4 | ALT4 | ||
ALT5 | ALT5 | ||
ALT6 | ALT6 | ||
ALT7 | ALT7 |
Well | Volume | Reagent |
ALT1 - ALT7 | 50 µL | Serial Dilutions (15.6 to 1000 mU/mL) |
BL | 50 µL | DPBS with 0.1% BSA |
TS | 50 µL | test sample |
- Prepare ALT standards (ALT), 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.
Note Dilute the test samples to the concentration range in DPBS buffer with 0.1% BSA if needed. - Add 50 µL of ALT working solution to 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 plate, add 25 µL of ALT working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at 37°C for 60 min to 120 minutes, protected from light.
Note The background of Blank Control increases with time. - Monitor the absorbance increase with an absorbance plate reader at the absorbance ratio of A570nm/A610nm.
Images
Figure 1. ALT dose response was measured with Amplite Colorimetric Alanine Aminotransferase Assay Kit in a 96-well white, clear-bottom plate using a SpectraMax microplate reader (Molecular Devices).
Figure 2. Analysis of serum transaminases following injection with viruses.
(A) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (B) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. (C) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (D) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. Viruses were injected with a dose of 1.0X108 virus particles / kg of body weight. 20μL of PBS was injected as a vehicle control. The sham control group did not undergo any surgical procedure. Serum samples were collected 6h, 3, 7 or 30 days post-injection of viruses or PBS. All data represent the means ± SD of three mice. Source: 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, by Faizule Hassan et al., PLOS, Feb. 2018
(A) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (B) ALT concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. (C) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the liver. (D) AST concentration following injection with AdEasy, AdEasy-HMGA-6 or PBS as vehicle control into the pancreas. Viruses were injected with a dose of 1.0X108 virus particles / kg of body weight. 20μL of PBS was injected as a vehicle control. The sham control group did not undergo any surgical procedure. Serum samples were collected 6h, 3, 7 or 30 days post-injection of viruses or PBS. All data represent the means ± SD of three mice. Source: 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, by Faizule Hassan et al., PLOS, Feb. 2018
Citations
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Journal: Pharmaceutics (2022): 1108
Pharmacologic IRE1/XBP1s activation promotes systemic adaptive remodeling in obesity
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Safety of nanoparticles based on albumin-polymer conjugates as a carrier of nucleotides for pancreatic cancer therapy
Authors: Taguchi, Kazuaki and Lu, Hongxu and Jiang, Yanyan and Hung, Tzong-Tyng and Stenzel, Martina Heide
Journal: Journal of Materials Chemistry B (2018)
Authors: Taguchi, Kazuaki and Lu, Hongxu and Jiang, Yanyan and Hung, Tzong-Tyng and Stenzel, Martina Heide
Journal: Journal of Materials Chemistry B (2018)
Reparative effects of astaxanthin-hyaluronan nanoaggregates against retrorsine-CCl4-induced liver fibrosis and necrosis
Authors: Wu, Yi Jhen and Wu, Yu Chiuan and Chen, I-Fen and Wu, Yi-Lung and Chuang, Chin Wen and Huang, Han Hsiang and Kuo, Shyh Ming
Journal: Molecules (2018): 726
Authors: Wu, Yi Jhen and Wu, Yu Chiuan and Chen, I-Fen and Wu, Yi-Lung and Chuang, Chin Wen and Huang, Han Hsiang and Kuo, Shyh Ming
Journal: Molecules (2018): 726
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
Authors: Hassan, Faizule and Lossie, Sarah L and Kasik, Ellen P and Channon, Audrey M and Ni, Shuisong and Kennedy, Michael A
Journal: PloS one (2018): e0192882
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Journal: PloS one (2018): e0192882
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FAQ
Why should I use an absorbance ratio at A575nm/A605nm when using most of your Amplite® Colorimetric Assay Kits?
How should I reconstitute an NADPH standard?
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Will Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence* work with NADP/NADPH? Can this kit measure NADP+ and NADPH?
What is the concentration of calcium inside cells?
What assay kits measure NADP/NADPH from cell samples?