Simple, direct and automation-ready procedures for measuring AST activity find wide applications in research and drug discovery. 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.

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.

Direct Assays: AST activity in serum, plasma and other biological samples. Drug Discovery/Pharmacology: effects of drugs on AST activity.

Assay Buffer: 24ml Cofactor: 120ul Enzyme Mix: 120ul NADH: 500ul Storage conditions. The kit is shipped on ice. Store all reagents at 20°C. Shelf life of six months after receipt. Precautions: Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Equilibrate all components to room temperature. Keep thawed enzymes 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). Samples should be clear and free of particles or precipitates. Hemolyzed samples should not be used. Procedure using 96-well plate 1. Samples and controls. Transfer 20ul sample to each well. For each assay plate, include two wells with 20ul distilled water to be used for the NADH Standard and Blank. Keep plate at the desired temperature (e.g. 37°C). 2. Prepare Working Reagent for Sample and Standard wells, by mixing for each well, 200ul Assay Buffer, 1ul Cofactor, 1ul Enzyme Mix and 4ul NADH. Warm to desired temperature (e.g. 37°C). Prepare Blank Reagent for the Blank well, by mixing 200ul Assay Buffer, 1ul Cofactor, 1ul Enzyme Mix and 4ul H2O. Warm to desired temperature (e.g. 37°C). 3. Add 200ul Working Reagent to the Standard and Sample wells, and 200ul Blank Reagent to the Blank well. Immediately tap plate to mix, incubate at the desired temperature and read OD340nm at 5 min and at 10 min. Alternatively, record kinetics at 340 nm.

Procedure using cuvettes 1. For each assay, include one Standard and one Blank control. For each Sample and Standard, prepare Working Reagent by mixing 1000ul Assay Buffer, 5ul Cofactor, 5ul Enzyme Mix and 20ul NADH. Transfer 990ul Working Reagent to each sample cuvette and standard cuvette. Warm to desired temperature (e.g. 37°C). To Blank control cuvette, add 960ul Assay Buffer, 5ul Cofactor, 5ul Enzyme Mix and 20ul H2O. Warm to desired temperature (e.g. 37°C). 2. Prewarm sample to the desired temperature. Add 100ul Sample to the Sample Cuvette. Transfer 100ul H2O to the Standard cuvette and to Blank Control cuvette, respectively. 3. Mix immediately. Read OD340nm at 5 min and 10 min. Alternatively, record kinetics at 340 nm.

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 (OD5 min-OD10min). Determine AST activity using the following equation, AST = DODS-DODNADH ODSTD–ODBLK 388 x (U/L) ODSTD and ODBLK are the OD340nm values of NADH Standard and Blank at 5 min, respectively. The factor 388 is derived from Factor = 4ul Vol.NADH 206ul Vol.WR 10mM NADH x x 200ul Vol.WR 220ul Vol.Total x 11 (sample dilution) 5 min = 388uM/min If the calculated AST activity is higher than 100 U/L, dilute sample in Assay Buffer and repeat assay. Multiple results by the dilution factor.

Pipeting devices and accessories. Clear bottom 96-well plates (e.g. Corning Costar) and plate reader or spectrophotometer and cuvettes for measuring OD340nm.