Application

This kit can measure Aspartate Aminotransferase (AST/GOT) activity in animal serum (plasma), tissue, culture cells and cell culture supernatant, etc.

Detection significance

AST/GOT is a key enzyme in nitrogen metabolism, which is widely found in plasma and body tissues, including liver, heart, skeletal muscle, kidney, brain, pancreas, lung and erythrocyte. Changes in AST/GOT activity were found in acute pancreatitis, ischemic stroke, severe burns, periodontitis, acute renal disease and motor neuron disease.

Detection principle

AST/GOT enables alpha-ketoglutaric acid and aspartic acid to displace amino and keto groups to form glutamic acid and oxaloacetic acid. Oxaloacetic acid can decarboxylate itself to form Pyroracemic acid during the reaction. Pyroracemic acid reacted with 2,4-dinitrophenylhydrazine(DNPH) to form 2,4, dinitrophenylhydrazone showing reddish brown in alkaline solution. Measure the OD values and calculate the enzyme activity.

Experimental instruments

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 5 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 1.1 IU/L.

2. The intra-assay CV is 5.3% and the inter-assay CV is 6.8%.

3. The detection range of the kit is 1.1-72.3 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.