Description
Alanine Transaminase Assay Kit (BA0074) (BA0074)
The Alanine Transaminase Assay Kit (SKU: BA0074) provides a simple, direct and automation-ready procedure for measuring alanine transaminase (ALT) activity. ALT, also known as serum alanine aminotransferase or SGPT, facilitates the conversion of alanine and alpha-ketoglutarate to pyruvate and glutamate and plays an important role in gluconeogenesis and amino acid metabolism. It is found mainly in the liver, and increased serum ALT activity is widely used as a marker for liver damage. This assay is based on the quantification of the pyruvate produced by ALT: pyruvate and NADH are converted to lactate and NAD by lactate dehydrogenase, and the decrease in NADH absorbance at 340 nm is proportional to ALT activity. The convenient assay can be carried out in a microplate or a cuvette and takes only 10 minutes.
| Product Name: | Alanine Transaminase Assay Kit (BA0074) |
| SKU: | BA0074 |
| Detection Method: | Colorimetric (kinetic) |
| Detection Range: | 2 to 100 U/L |
| Sample Type: | Serum, plasma and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 10 minutes (readings at 5 and 10 minutes) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt; 3 weeks after reconstitution |
| Shipping: | Gel Pack |
Colorimetric kinetic determination of alanine transaminase activity. Pyruvate produced by ALT is coupled to lactate dehydrogenase, and the decrease in NADH absorbance at 340 nm is proportional to ALT activity.
- Sensitive with a linear detection range of 2 to 100 U/L
- Simple and convenient
- Can be performed in a microplate or a cuvette
- Takes only 10 minutes
- Direct assays of ALT activity in serum, plasma and other biological samples
- Drug discovery and pharmacology: effects of drugs on ALT activity
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Preparation. Equilibrate all components to room temperature. Reconstitute the NADH Reagent with 1000 uL distilled water (final 10 mM); reconstituted reagent is stable for three weeks at -20C. Mix Assay Buffer well and keep thawed enzyme on ice. Assays can be performed at 37C or room temperature. |
| 2 | Samples and controls. Transfer 20 uL of each sample to separate wells. For each plate include two wells with 20 uL distilled water for the NADH Standard and Blank. Keep the plate at the desired temperature. |
| 3 | Working reagent. For each sample and standard well mix 200 uL Assay Buffer, 5 uL Cosubstrate, 1 uL LDH and 4 uL reconstituted NADH; for the Blank use 4 uL distilled water instead of NADH. Warm to the desired temperature. |
| 4 | Add 200 uL working reagent to each sample and standard well and 200 uL blank reagent to the blank well. Tap to mix, then read OD at 340 nm at 5 minutes and 10 minutes, or record kinetics at 340 nm. |
For each sample calculate the change in NADH (OD at 5 minutes minus OD at 10 minutes) and similarly for the NADH standard. ALT = ((change in ODsample - change in ODNADH) / (ODstandard - ODblank)) x 381 U/L, where 381 uM/min is the derived factor. If activity exceeds 100 U/L, dilute in Assay Buffer, repeat and multiply by the dilution factor. One unit of ALT catalyses the conversion of 1 umole of alanine to pyruvate per minute at pH 7.7.
| Component | Quantity | Storage |
| Assay Buffer | 24 mL | -20C |
| Cosubstrate | 600 uL | -20C |
| LDH | 120 uL | -20C |
| NADH Reagent (dried) | 1 vial | -20C |
