bSLSBio LifeSpan BioSciences, Inc. Aspartate Transaminase (AST) Assay Kit (Colorimetric) LS-K297-100 (100 Tests) • Store at -20°C Introduction Aspartate Transaminase (AST), also known as serum glutamic oxaloacetic transaminase (GOT) or aspartate aminotransferase (ASAT/AAT), facilitates the conversion of aspartate and a-ketoglutarate to oxaloacetate and glutamate. There are two isoenzymes in humans: GOT1 is a cytosolic isoenzyme derived from red blood cells and heart; GOT2 is the mitochondrial isoenzyme found mainly in the liver. AST is elevated in liver and muscle diseases. It is part of diagnostic tests for liver function, myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease and trauma. Simple, direct and automation-ready procedures for measuring AST activity find wide applications in research and drug discovery. LSBio's' AST activity assay is based on the quantification of oxaloacetate produced by AST. In this assay, oxaloacetate and NADH are converted to malate and NAD by the enzyme malate dehydrogenase. The decrease in NADH absorbance at 340 nm is proportionate to AST 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: AST activity in serum, plasma and other biological samples. • Drug Discovery/Pharmacology: effects of drugs on AST 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]

Aspartate Transaminase (AST) Assay Kit (Colorimetric) LS-K297-100 (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)....

bSLSBio LifeSpan BioSciences, Inc. Aspartate Transaminase (AST) Assay Kit (Colorimetric) LS-K297-100 (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 (AODs). Similarly, calculate the rate (AODnadh) for the NADH standard (OD5min - OD10min). Determine AST activity using the following equation, AODs - AODnadh (U/L) ODstd — ODblk ODstd and ODblk are the OD340nm values of NADH Standard and Blank at 5 min, respectively. The factor 388 is derived from ^ llllinil4 pL Vol.NADH 200 pL Vol.WR 11 (sample...