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Alanine Transaminase Activity Assay Kit (Colorimetric/Fluorometric) (ab105134) is a rapid and simple assay where alanine transminase (ALT) catalyzes the transfer of an amino group from alanine to a-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate. The pyruvate is detected in a reaction that concomitantly converts a nearly colorless probe to both color (ODmax = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The kit provides a rapid, simple, sensitive, and reliable test suitable for high throughput activity assay of ALT with a detection limit of 10 mU per well.
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Alanine transaminase, also called alanine aminotransferase or serum glutamic pyruvic transaminase (ALT, ALAT, SGPT, EC 220.127.116.11) is a transaminase enzyme found in serum and in various bodily tissues, but usually associated with the liver. It catalyzes the reaction: a-ketoglutarate + alanine ? glutamate + pyruvate. It is commonly measured clinically as a part of a diagnostic liver function test to determine liver health. Diagnostically, it is almost always measured in units/liter (U/L).
|ALT Positive Control (lyophilized)||Blue||1 vial|
|ALT Substrate (lyophilized)||Orange||1 vial|
|Pyruvate Standard (100 nmol/µl)||Yellow||1 x 100µl|
|ALT Assay Buffer||WM||1 x 25ml|
|ALT Enzyme Mix (lyophilized)||Green||1 vial|
|OxiRed™ (in DMSO||Red||1 x 200µl|
Our Abpromise guarantee covers the use of ab105134 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent dilution.|
Liver samples from high fat diet (HFD) and standard carbohydrate diet (CHD) BALB/c and C57BL6/J mice were homogenised in an ALT assay buffer for the determination of ALT activity using ab105134. A separate batch of liver extracts was prepared and incubated in a buffer containing NP40 (5%) and supernatants containing the triglycerides were separated. Triglycerides concentration was determined on the supernatant fraction using ab65336. ALT activity and triglycerides concentration were determined by measuring OD at 570nm.
Fluorometric standard curve: mean of duplicates (+/- SD) with background reads subtracted.
Alanine transaminase measured in mouse tissue lysates showing quantity (mU) per mg of tested sample.
Protein concentration for samples varied from 4 mg/mL to 13 mg/mL. Samples were diluted 9-27 fold and measured colorimetrically.
Pyruvate measured colorimetrically in cell lysate after 20 min and 40 min incubation time showing quantity (nmol) per 1 mln of tested cells.
Measurement of alanine transaminase in HepG2 cells (10 μg) and liver lysate (15 μg).
Pyruvate measured fluorometrically in cell lysate after 20 min and 40 min incubation time showing quantity (nmol) per 1 mln of tested cells.
Pyruvate measured in biological fluids after 20 min and 40 min incubation time showing quantity (nmol) per ml of tested sample.