Alanine Aminotransferase (ALT/GPT) Activity Assay Kit

K097

96T(42 samples)

0.75-72.30IU/L

0.75IU/L

The kit should be stored at 2-8℃ shading light for 6 months.

Alanine aminotransferase (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°C. 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.

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

1.Reagent preparation

a.Bring all reagents to room temperature before use.

b.The preparation of alkali working solution：

Mix alkali reagent and double distilled water thoroughly at the volume ratio of 1:9. The alkali working solution should be prepared on spot.

c.Take part of substrate solution, and incubate it at 37°C for 10 min.

2.Sample preparation

a.Preparation of sample

Serum and plasma: detect directly.

Urine: collect fresh urine and centrifuge at 10000 g for 15 min at 4°C. Take the supernatant and preserve it on ice for detection.

Tissue sample:

① Harvest the amount of tissue needed for each assay (initial recommendation 20 mg).

② Wash tissue in cold PBS (0.01 M, pH 7.4).

③ Homogenize 20 mg tissue in 180 µL PBS (0.01 M, pH 7.4) with a dounce homogenizer at 4°C.

④ Centrifuge at 10000×g for 10 minutes to remove insoluble material. Collect supernatant and keep it on ice for detection.

⑤ Meanwhile, determine the protein concentration of supernatant (K001).

Cells:

① Harvest the number of cells needed for each assay (initial recommendation 1×106 cells).

② Wash cells with PBS (0.01 M, pH 7.4).

③ Homogenize 1×106 cells in 300 µL PBS (0.01M, pH 7.4) with a ultrasonic cell disruptor at 4°C.

④ Centrifuge at 10000×g for 10 minutes to remove insoluble material. Collect supernatant and keep it on ice for detection.

⑤ Meanwhile, determine the protein concentration of supernatant (K001).

b.Dilution of sample

Before the formal test, 2-3 samples with significant differences should be selected and diluted into different concentrations for pre-test. Determine whether dilution is required according to the pre-test results and the kit detection range of 0.75-72.30 IU/L. The recommended dilution factor for different samples is as follows (for reference only):

Note: The diluent is saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

3.Add samples and test

① Standard wells: Add 5 μL of buffer solution to the standard wells respectively (multi-channel pipette is recommended to be used). Add 20, 18, 16, 14, 12, 10 μL of substrate solution to the standard wells from A to F, respectively. Add 0, 2, 4, 6, 8, 10 μL of 2 mmol/L sodium pyruvate to the standard wells from A to F, respectively.

Sample wells: Add 20 μL of substrate solution (pre-heated at 37°C for 10 min) and 5 μL of sample.

Control wells: Add 20 μL of substrate solution (pre-heated at 37°C for 10 min).

② Mix well (this is very important), then incubate at 37°C for 30 min.

③ Add 20 μL of chromogenic agent to each well.

④ Control wells: Add 5 μL of sample to control wells.

⑤ Mix well with microplate reader for 10 s, incubate at 37°C for 20 min.

⑥ Add 200 μL of alkali working solution to each well (the multi-channel pipette is recommended).

⑦ Mix fully with microplate reader for 10 s, stand for 15 min at room temperature and measure the OD value of each well with microplate reader at 510 nm.

the standard curve: y = a x² + b x + c

Definition of Carmen unit: 1 mL of sample, the total volume of reaction is 3 mL, wavelength is 340 nm, optical path is 1 cm, react at 25°C for 1 min, the amount of generated pyruvic acid which oxidize NADH to NAD+ and cause absorbance decreasing 0.001 is as 1 unit. (1 Carmen unit = 0.482 IU/L, 25°C)

Definition of international unit: The enzyme amount of 1 μmol of NADH consumed in reaction system (1 mL sample or 1 g tissue protein, 25°C) per minute is defined as 1 unit (wavelength is 340 nm, optical path is 1 cm).

The sample:

1. Serum (plasma) sample:

ALT/GPT activity (IU/L) = [a × (ΔA510) ² + b × ΔA510+ c] × 0.482 × f

2. Tissue and cells sample:

ALT/GPT activity (IU/gprot) = [a × (ΔA510) ² + b × ΔA510+ c] × 0.482 × f ÷ Cpr

Note:

ΔA510: ODsample-ODcontrol

f: Dilution factor of sample before tested.

Cpr: Concentration of protein in sample (gprot/L).

1.This assay kit is for Research Use Only. We will not response for any arising problems or legal responsibilities causing by using the kit for clinical diagnosis or other purpose.

2.Please read the instructions carefully and adjust the instruments before the experiments. Please follow the instructions strictly during the experiments.

3.Protection methods must be taken by wearing a lab coat and latex gloves during the experiment.

4.The detection range of the kit is not equivalent to the concentration range of the substance to be measured in the sample. If the concentration of substance is not within the detection range exactly, an extra dilution or concentration should be taken for the sample.

5.If the samples to be tested are not among the types listed in the instructions, it is recommended to conduct a preliminary experiment to verify the effectiveness of the test.

6.The experimental results are closely related to the situation of reagents, operations, environment and so on. FineTest will guarantee the quality of the kits only, and NOT be responsible for the sample consumption caused by using the assay kits. It is better to calculate the possible usage of sample and reserve sufficient samples before use.