Aspartate Aminotransferase (AST/GOT) Colorimetric Assay Kit

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

      Catalog number:E-BC-K236-M

      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.

      Add to cart Compare Bulk request Manual

      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.


       

      A

      B

      C

      D

      E

      Reagent 1 (μL)

      5

      5

      5

      5

      5

      Reagent 3 (μL)

      20

      18

      16

      14

      12

      Reagent 2 (μL)

      0

      2

      4

      6

      8

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

      Reagent 4 (μL)

      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

      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 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.



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      Verified Customer

      a*****hSubmitted [ Jun 05 2019 ]

      • Application:Biochemical
      • Description:the biggest advantage of this kit is the small sample size.on the other hand,no special drawing tools are required,we can draw a standard curve in excel,which is convenient.
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