bSLSBio LifeSpan BioSciences, Inc. Alanine Transaminase (ALT) Assay Kit (Colorimetric) LS-K308-100 (100 Tests) • Store at -20°C Introduction Alanine Transaminase (ALT), also known as serum alanine aminotransferase (ALAT) or pyruvic transaminase (SG PT), facilitates the conversion of alanine and a-ketoglutarate to pyruvate and glutamate. ALT plays an important role in gluconeogenesis and amino acid metabolism. 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 increased serum ALT activity is widely used as a marker for liver damage. Simple, direct and automation-ready procedures for measuring ALT activity find wide applications in research and drug discovery. LSBio's ALT activity assay is based on the quantification of pyruvate produced by ALT. In this assay, pyruvate and NADH are converted to lactate and NAD by the enzyme lactate dehydrogenase (LDH). The decrease in NADH absorbance at 340 nm is proportion al to ALT activity. Key Features • Sensitive. Linear detection range: 2-100 U/L. • Simple and convenient. This simple, convenient assay can be carried out in a microplate or a cuvette and takes only 10 min. Applications • Direct Assays: ALT activity in serum, plasma and other biological samples. • Drug Discovery/Pharmacology: effects of drugs on ALT activity. Materials Not Supplied Pipetting devices and accessories. Clear bottom 96-well plates (e.g. Corning Costar) and plate reader or spectrophotometer and cuvettes for measuring OD340nm. Storage The kit is shipped on ice. Store all kit components at -20 °C. FOR RESEARCH USE ONLY! Not for use in humans. LifeSpan BioSciences, Inc. • 2401 Fourth Avenue, Suite 900, Seattle, WA 98121 www.LSBio.com • (206) 464-1554 • [email protected]

Alanine Transaminase (ALT) Assay Kit (Colorimetric) LS-K308-100 (100 Tests) • Store at -20°C Assay Procedure Equilibrate all components to room temperature. Reconstitute the NADH Reagent tube with 1000 μL dH2O (final 10 mM). Unused reconstituted NADH reagent is stable for three weeks when stored frozen at -20°C. Mix assay buffer well by vigorous shaking. Keep thawed enzyme on ice. Assays can be performed at 37°C or at room temperature. Prior to assay, bring the working reagents, microplate and spectrophotometer to the desired temperature. Assay is compatible with serum or plasma (heparin, EDTA)....

Alanine Transaminase (ALT) Assay Kit (Colorimetric) LS-K308-100 (100 Tests) • Store at -20°C Calculations For each Sample, calculate the rate of NADH consumption by subtracting the OD at 10 min from the OD at 5 min (DODS). Similarly, calculate the rate (DODNADH) for the NADH standard (OD5min - OD10min). Determine ALT activity using the following equation, ODSTD and ODBLK are the OD340nm values of NADH Standard and Blank at 5 min, respectively. The factor 381 is derived from If the calculated ALT activity is higher than 100 U/L, dilute sample in Assay Buffer and repeat assay. Multiply results...