| Alanine Transaminase (ALT), also known as serum alanine aminotransferase (ALAT) or pyruvic transaminase (SGPT), 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. |
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| Catalog # | A1210-23 |
| Simple, direct and automation-ready procedures for measuring ALT activity find wide applications in research and drug discovery. 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 proportional 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. |
| Kit Contents | Assay Buffer: 24ml, LDH: 120ul Cosubstrate: 600ul NADH: 500ul |
| Storage conditions. The kit is shipped on ice. Store all reagents at -20°C. Shelf life of three 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. |
| Assay Procedures | Equilibrate all components to room temperature. 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). 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 of each sample to separate wells. Also, 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, 5ul Cosubstrate, 1ul LDH and 4ul NADH. Warm to desired temperature (e.g. 37°C). |
| Prepare Blank Reagent for the Blank well by mixing: 200ul Assay Buffer, 5ul Cosubstrate, 1ul LDH and 4ul H2O. Warm to desired temperature (e.g. 37°C). |
| 3. Add 200ul Working Reagent to each Sample and Standard well, 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, 25ul Cosubstrate, 5ul Enzyme Mix and 20ul NADH. Transfer 1000ul Working Reagent to each sample cuvette and standard cuvette. Warm to desired temperature (e.g. 37°C). For Blank control, mix 1000ul Assay Buffer, 25ul Cosubstrate, 5ul Enzyme Mix and 20ul H2O. Transfer 1000ul Reagent to the Blank control cuvette. 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. Mix immediately. 3. Read OD340nm at 5 min and 10 min. Alternatively, record kinetics at 340 nm. |
| Calculation | For each Sample, calculate the rate of NADH consumption by subtracting the OD at 10 min from the OD at 5 min ( ODS). Similarly, calculate the rate ( ODNADH) for the NADH standard (OD5min-OD10min). |
| Determine ALT activity using the following equation, |
| ALT=381 x |
| ODS- ODNADH |
| ODSTD–ODBLK |
| (U/L) |
| ODSTD and ODBLK are the OD340nm values of NADH Standard and Blank at 5 min, respectively. The factor 381 is derived from |
| Factor=10mM NADH x 4ul Vol.NADH x 200ul Vol.WR x 11 (sample dilution) |
| 210ul Vol.WR If the calculated ALT activity is higher than 100 U/L, dilute sample in |
| 5 min |
| 220ul Vol.Total |
| = 381uM/min Assay Buffer and repeat assay. Multiply results by the dilution factor. |
| Materials Required, But Not Provided | Pipeting devices and accessories. Clear bottom 96-well plates (e.g. Corning Costar) and plate reader or spectrophotometer and cuvettes for measuring OD340nm. |
| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
