Application

This kit can be used to measure ALT/GPT activity in animal serum (plasma), tissue, culture cells and cell culture supernatant, etc.

Detection significance

Alanine aminotransferase (ALT) is widely found in plasma and various tissues of the body, including liver, kidney, heart and skeletal muscle. ALT is an important pyridoxal phosphate dependent enzyme in the intermediate metabolism of glucose and protein. Clinically, the activity of serum alanine aminotransferase is often used as a marker for alcoholic liver disease, liver cirrhosis and acute viral hepatitis.

Detection principle

ALT catalyze the amino conversion reaction between alanine and α-ketoglutaric acid to produce pyruvic acid and glutamic acid at pH 7.4 and 37℃. Then phenylhydrazine was added to form phenylhydrazone with pyruvic acid. Phenylhydrazone is reddish brown under alkaline conditions. ALT activity can be calculated by measuring the OD values at 510 nm.

Experimental instrument

Test tube, Micropipettor, 37℃water bath, Vortex mixer, Microplate reader (510 nm)

Sample preparation

1. Serum (plasma) and other liquid sample: Detect the sample directly.

2. Animal tissue sample: Accurately weigh the tissue sample, add 9 times the volume of PBS (0.01 M, pH7~7.4) according to the ratio of Weight (g): Volume (mL) =1:9. Mechanical homogenate the sample in ice water bath. Centrifuge at 10000 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

3. Culture cell sample: Wash the cells with PBS (0.01 M, pH7~7.4) for 1~2 times. Centrifuge at 1000 g for 10 min and then discard the supernatant and keep the cell sediment. Add PBS at a ratio of cell number (106): PBS (μL) =1: 300-500. Sonicate or grind with hand-operated in ice water bath. Centrifuge at 10000 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1. The preparation of standard curve

Set 6 wells of micro-plate for standard and operate according to the following operating table.

2. The measurement of samples

Note: Steps 1 and 2 can be progress at the same time.

Technical parameter

1. The sensitivity of the kit is 0.75 IU/L.

2. The intra-assay CV is 5.3% and the inter-assay CV is 9%.

3. The detection range of the kit is 0.75-72.3 IU/L.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.

Citations