Glutathione Peroxidase (GSH-PX) Colorimetric Assay Kit

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

      Catalog number:E-BC-K096-S

      Size:
      • 50 Assays
      • 100 Assays
      Qty:
      - +
      Price: $180

      Detection method: Colorimetric method

      Detection instrument: Spectrophotometry (Visible range)

      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 be used to measure GSH-PX activity of animal serum, plasma, tissue, cells, cell culture supernatant and other samples.

       

      Detection significance

      Glutathione Peroxidase (GSH-PX) is an important enzyme that catalyzes decomposition of hydrogen peroxide. GSH specifically catalyze the reaction between GSH and hydrogen peroxide, protecting cell membrane structure and keeping membrane function integrity. Se-cysteine is the active center of the GSH-PX. Determination of GSH-PX activity in organism can be an indicator of selenium level as Se is essential section of GSH-PX.

       

      Detection principle

      Glutathione Peroxidase (GSH-PX) can promote the reaction of hydrogen peroxide (H2O2) and reduced glutathione to produce H2O and oxidized glutathione (GSSG). The activity of glutathione peroxidase can be expressed by the rate of enzymatic reaction. The activity of glutathione can be calculated by measuring the consumption of reduced glutathione. Hydrogen peroxide (H2O2) and reduced glutathione can react without catalysis of GSH-PX, so the portion of GSH reduction by non-enzymatic reaction should be subtracted. GSH can react with dinitrobenzoic acid to produce 5-thio-dinitrobenzoic acid anion, which showed a stable yellow color. Measure the absorbance at 412nm, and calculate the amount of GSH.

                                                                          

      Experimental instrument

      Test tube

      Micropipettor

      Vortex mixer

      Centrifuge

      37 water bath/gas bath

      Spectrophotometer (412 nm)

       

      Sample preparation

      1.   Serum (Plasma):

      Detect the sample directly. If the concentration is beyond the linear range, then dilute the sample with normal saline before detection.

       

      2.   10% tissue homogenate:

      Accurately weigh the tissue weight, add 9 times the volume of homogenized medium 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 for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

       

      3.   Cell sample:

      Collect the 1×106 cells and centrifuge at 1000~1500 g for 10 min. Discard the supernatant and keep the cell sediment. Add 300~500 μL of PBS (0.01 M, pH 7.4) to prepare cell suspension. Sonicate in ice water bath (power: 300 W, 3~5 second/time, interval for 30 seconds, repeat for 3~5 times) or grind with hand-operated. Centrifuge at 10000 g for 10 min, then take the supernatant for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).


       

      Pre-experiment

      1.    Determine optimal dilution factor of samples before formal experiment. Samples are diluted into difference concentration with ddH2O, then take the pre-experiment according to the operation procedures. Calculate the inhibition ratio of serial diluent, and choose the optimal dilution factor when inhibition ratio in the range of 45%~55%.   


      2.    The optimal dilution factor of sample are different for different species, the GSH-PX also are different for different samples. So it is best to do a preliminary experiment to determining optimal dilution factor for a new sample.

       

      3.    It is best to reserve 3 paralleled tubes with different sampling volumes in preliminary experiment for determining the optimal sampling volume. For example,

      Tissue homogenate: Pipet 200 μL of 5%, 1%, 0.5%, 0.4%, 0.3%, 0.1% tissue homogenate supernatant respectively for the assay.

      Serum: Dilute serum samples with normal saline at ratio of 1:1, 1:4, 1:7, 1:9 respectively. Then pipet 100 μL of undiluted and diluted serum samples for the assay.

       

      4.    Adjust dilution factor: If inhibition ratio > 60%, need to further dilute the sample then process the test. If inhibition ratio < 10%, need to increase sample concentration.

       

      Operation steps

      1. For tissue, cells and cell culture supernatant sample:

      1) Enzymatic reaction (Reagent 1 application solution are pre-warmed in advance at 37)

       

      Non-enzyme tube

      Enzyme tube

      1 mmol/L GSH (mL)

      0.2

      0.2

      Sample (mL)

       

      0.2

      Pre-heat the tubes at 37 water bath for 5 min. Preheat Reagent 1 application solution at 37 for 5 min at the same time.

      Reagent 1 application solution (mL)

      0.1

      0.1

      React at 37 water bath for 5 min accurately.

      Reagent 2 (mL)

      2

      2

      Sample (mL)

      0.2

       

      Mix fully and centrifuge at 3100 g for 10 min, then take 1 mL of supernatant for chromogenic reaction.


       

      2) Chromogenic reaction

       

      Blank tube

      Standard tube

      Non-enzyme tube

      Enzyme tube

      GSH standard application solution (mL)

      1

       

       

       

      20 μmol/L GSH standard solution (mL)

       

      1

       

       

      Supernatant (mL)

       

       

      1

      1

      Reagent 3 application solution (mL)

      1

      1

      1

      1

      Reagent 4 (mL)

      0.25

      0.25

      0.25

      0.25

      Reagent 5 application solution (mL)

      0.05

      0.05

      0.05

      0.05

           Mix fully and stand for 15 min at room temperature. Set to zero with double-distilled water and measure the OD values of each tube at 412 nm with 1 cm diameter cuvette.

       

      2.  For serum/plasma sample:

      1) Enzymatic reaction (Reagent 1 application solution are pre-warmed in advance at 37)

       

      Non-enzyme tube

      Enzyme tube

      1mmol/L GSH (mL)

      0.2

      0.2

      Sample (mL)

       

      0.1

      Pre-heat the tubes at 37 water bath for 5 min. Preheat Reagent 1 application solution at 37 for 5 min at the same time.

      Reagent 1 application solution (mL)

      0.1

      0.1

      React at 37 water bath for 5 min accurately.

      Reagent 2 (mL)

      2

      2

      Sample (mL)

      0.1

       

      Mix fully and centrifuge at 3100 g for 10 min, then take 1 mL of supernatant for chromogenic reaction.

       

      2) Chromogenic reaction


       

      Blank tube

      Standard tube

      Non-enzyme tube

      Enzyme tube

      GSH standard application solution (mL)

      1

       

       

       

      20 μmol/L GSH standard solution (mL)

       

      1

       

       

      Supernatant (mL)

       

       

      1

      1

      Reagent 3 application solution (mL)

      1

      1

      1

      1

      Reagent 4 (mL)

      0.25

      0.25

      0.25

      0.25

      Reagent 5 application solution (mL)

      0.05

      0.05

      0.05

      0.05

      Mix fully and stand for 15 min at room temperature. Set to zero with double-distilled water and measure the OD values of each tube at 412 nm with 1 cm diameter cuvette.

        

      Technical parameter

      1.  The sensitivity of the kit is 12.65 U.

      2.  The intra-assay CV is 4.9% and the inter-assay CV is 9.3%.

      3.  The recovery of the kit is 105%.

      4.  The detection range of the kit is 12.65-387 U.


      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

      1. Publication: Kim J, Choung S Y. Pinus Densiflora Bark Extract Prevents Selenite-induced Cataract Formation In The Lens Of Sprague Dawley Rat Pups[J]. Molecular Vision, 2017, 23: 638-648.
        Sample Type: Tissue homogenate
      2. Publication: Siboto A, Sibiya N, Khathi A, et al. The Effects of Momordica balsamina Methanolic Extract on Kidney Function in STZ-Induced Diabetic Rats: Effects on Selected Metabolic Markers[J]. Journal of Diabetes Research, 2018, 2018.
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      • Reviews (0)
      • Q&A (1)
      Q

      T****************gSubmitted [ Apr 23 2019 ]

      Asked: We don\'t have 412 nm of colorimetric spectro filter in my lab, we only have the 415 nm. And so, is it ok if we use 415nm? would it effect the accuracy of the measurement significantly? Could give me the contract of distributor in Thailand. Thank you.

      Reply

      A

      adminSubmitted [ Apr 23 2019 ]

      Answered: Dear Thapanee, Thanks for your message. It is ok to use 412nm to test the kit. Pls contact with the distributor to have the kit. Pacific Science Co.,Ltd. 62,64 Soi Charansanitwong 49/1 Charansanitwong rd. Bangbumru Bangplad Bangkok 10700 THAILAND Tel : +662-4330068-9 ext. 212 Email : pacscien@ksc.th.com Any questions, pls feel free to ask. Best regards,

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