Alanine Aminotraasferase (ALT/GPT) Colorimetric Assay Kit

    • Biochemical Assay Kit-Elabscience
    <
    >
    • Biochemical Assay Kit-Elabscience
    • Biochemical Assay Kit-Elabscience

      Catalog number:E-BC-K235

      Size:
      • 48T
      • 96T
      Qty:
      - +
      Price: $120

      Detection method: Colorimetric method

      Detection instrument: Microplate reader

      Valid period: 6 months

      Lead Time: 7~10 daysWelcome to order from local distributors.

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      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.


       

      A

      B

      C

      D

      E

      F

      Reagent 1 (μL)

      5

      5

      5

      5

      5

      5

      Reagent 3 (μL)

      20

      18

      16

      14

      12

      10

      Reagent 2 (μL)

      0

      2

      4

      6

      8

      10

      Mix fully (this is very important), then incubate at 37 for 30 min.

      Reagent 4 (μL)

      20

      20

      20

      20

      20

      20

      Mix fully with microplate reader for 10 s and incubate at 37 for 20 min.

      Reagent 5 working solution (μL)

      200

      200

      200

      200

      200

      200

      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.

      2. The measurement of samples

       

      Control well

      Sample well

      Reagent 3 (μL) (pre-heated at 37 for 10 min)

      20

      20

      Sample (μL)

       

      5

      Mix fully (this is very important), then incubate at 37 for 30 min.

      Reagent 4 (μL)

      20

      20

      Sample (μL)

      5

       

      Mix fully with microplate reader for 10 s and incubate at 37 for 20 min.

      Reagent 5 working solution (μL)

      200

      200

      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.

      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.



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      Verified Customer

      a****gSubmitted [ May 29 2019 ]

      • Application:Biochemical
      • Description:This alanine aminotransferase assay kit requires a small sample size, which is of great help to our real project.
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