Alanine transaminase (ALT), also known as alanine aminotransferase (ALAT) or serum glutamic pyruvic transaminase (sGPT), is a homodimeric enzyme dependent on cytoplasmic pyridoxal phosphate. It plays a vital role in cellular nitrogen metabolism, amino acid metabolism, and liver gluconeogenesis. ALT catalyzes the reversible transamination between alanine and α-ketoglutarate to form pyruvate and glutamate. ALT is found mainly in the liver, and, to a lesser extent, in kidney, heart, muscle, and pancreas tissues. Normal serum levels of ALT are low, and an elevation in serum ALT activity is a widely used marker for liver damage.

The RayBio® Alanine Transaminase Activity Assay Kit offers a user-friendly, reliable, and highly sensitive method for quantifying ALT activity in plasma, serum, cell lysates, and other biological fluid samples. In this assay, ALT catalyzes the transfer of the amino group from L-alanine to α-ketoglutarate resulting in the formation of pyruvate and L-glutamate. Simultaneously, lactate dehydrogenase catalyzes the reduction of pyruvate and the oxidation of NADH to NAD. The resultant rate of absorbance decrease is directly proportional to ALT activity.

• Microplate reader capable of measuring absorbance at 340 nm at 37°C
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Multi-channel pipettes to deliver 20 μl to 200 μl volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C
• 15 ml conical tubes

Positive control, sample and Sample Buffer (used as a blank) should be assayed in duplicate or triplicate. A freshly prepared positive control should be used each time the assay is performed.

1. Set up a microplate reader or a microplate incubator at 37°C.
2. Prepare Working Solution (See Reagent Preparation, section A), and incubate it at 37°C for at least five minutes.
3. Prepare Positive Control (See Reagent Preparation, section B).
4. Pipette 10 µL of sample, Sample Buffer (as the blank) and Positive Control into each well of the 96-well plate.
5. Transfer 100 µL of pre-warmed Working Solution into each well (it is recommended to use a multi-channel pipette), mix and incubate at 37°C for one minute.
6. After one minute, read and record absorbance at 340nm (A1 Reading). Repeat readings every minute for the next four minutes at 37°C. (A2, A3, A4, A5 Reading).