Glutathione Peroxidase (GSH-Px) Activity Assay Kit

K147

96T (40 samples)

34.34 U/mL

34.34–1036.64 U/mL

This assay kit is suitable for detecting GSH-Px activity in serum, plasma, cultured cells, culture media, and animal or plant tissue samples.

Store in the dark at 2–8 °C for 6 months.

Note: Reagents must be stored strictly in accordance with the storage conditions specified in the table above; reagents from different test kits must not be mixed. For small volumes of reagent, centrifuge the reagent prior to use to ensure that an adequate amount can be accurately measured.

GSH-Px catalyzes the reaction between hydrogen peroxide (H2O2) and reduced glutathione (GSH), yielding H2O and oxidized glutathione (GSSG). The enzymatic activity is quantified by measuring the rate at which the substrate GSH is oxidized per unit time; the residual GSH reacts with dithiodibinitrobenzoic acid (DTNB) to produce a yellow compound, the absorbance of which can be measured at 412 nm. Since GSH can also be oxidized in the absence of an enzyme (non-enzymatic reaction), the calculated enzymatic activity must be corrected for the portion of GSH depletion attributable to this non-enzymatic reaction.

When using this kit to analyze tissue and cell samples, it is necessary to determine the total protein concentration; we recommend using our own BCA kit (product no.: K001).

Microplate reader (400–420 nm, optimal detection wavelength: 412 nm), 37 °C incubator, vortex mixer, centrifuge

Double-distilled water, normal saline (0.9% NaCl), or PBS (0.01 M, pH 7.4)

Tip (1000 μL, 200 μL, 10 μL); EP tube (10 mL, 1.5 mL).

1. Before testing, equilibrate the reagents in the kit to room temperature (25°C).

2. Preparation of Reagent 1 Application Solution:

Prepare Reagent 1 by mixing double-distilled water in a 1:99 (v/v) ratio; prepare fresh solution each time, and the prepared Reagent 1 Application Solution shall be used within 8 hours.

3.Preparation of Reagent 6 Application Solution:

Mix Reagent 6 with double-distilled water in a 1:9 (v/v) ratio; if Reagent 6 solidifies into ice crystals, place it at 65 °C to allow it to dissolve. Any unused Reagent 6 Application Solution may be stored at 2–8 °C for up to 7 days.

4.Preparation of the 1 mmol/L GSH standard:

Add 10 mL of Reagent 6 Application Solution to one vial of Reagent 5, mix well, and aliquot the remaining portion; the aliquots can be stored at –20 °C for one month.

5. Preparation of the 100 μmol/L GSH standard solution:

Mix 1 mmol/L GSH solution with Reagent 6 Application Solution in a 1:9 (v/v) ratio, homogenize thoroughly, and store at 2–8 °C for 7 days.

6. Dilution of standard solutions at different concentrations:

1. Sample Processing

Sample requirements: The sample must not contain reducing agents such as DTT or 2-mercaptoethanol, nor detergents such as Triton X-100 or Tween.

Liquid samples, including serum and plasma: direct measurement.

Cell samples: Approximately 10^6 cells were added to 300–500 μL of PBS (0.01 M, pH 7.4) for homogenization. After homogenization, the mixture was centrifuged at 4 °C and 10,000 × g for 10 min; the supernatant was then transferred to ice and kept on ice for subsequent analysis. A portion of the supernatant was reserved for protein concentration determination.

Tissue samples: Homogenize tissue samples according to a ratio of tissue sample mass (g) to PBS (0.01 M, pH 7.4) (mL) of 1:9 (e.g., for 0.05 g of tissue sample, add 450 μL of PBS (0.01 M, pH 7.4) for homogenization); centrifuge at 4 °C at 10,000 × g for 10 min; collect the supernatant and place it on ice for subsequent analysis. Reserve a portion of the supernatant for protein concentration determination.

2.Calculation of sample inhibition rate

This assay kit can detect the GSH-Px inhibition rate within the range of 10%–50%, with an optimal inhibition rate range of 25%–45%; the corresponding concentration represents the optimal sampling concentration.

Inhibition rate calculation formula:

If the inhibition rate exceeds 50%, the sample concentration should be diluted before testing. If the inhibition rate is below 10%, the sample concentration should be increased, or the sample volume should be increased.

3. Sample dilution

Prior to formal testing, select 2–3 samples expected to exhibit significant differences and dilute them into various concentrations. Then, select the dilution factor for those samples that yield an inhibition rate between 25% and 45%, and proceed with formal batch testing. The dilution ratio ranges for the different samples are provided in the table below (for reference only):

Note: The dilution medium consists of normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

①For both non-enzymatic and enzymatic tubes, the supernatant after adding Reagent 2 and centrifugation must be clear.

②A preliminary experiment must be conducted after sample processing.

③Reagent 1 application solution must be prewarmed to 37 °C for 5 min.

④When the inhibition rate is less than 10%, the sample size may be increased.

Standard curve: y=ax+b

Calculation of results for serum, plasma, and culture media:

Definition: One unit of enzymatic activity is defined as the amount of enzyme required to reduce the GSH concentration in the reaction mixture by 1 μmol/L, after incubating 0.1 mL of serum (or plasma) at 37 °C for 5 min and correcting for non-enzymatic reactions.

Calculation of results for tissues and cells:

Definition: The enzyme activity is defined as the number of enzyme activity units per milligram of protein, determined by incubating the sample at 37 °C for 5 min, correcting for the effect of non-enzymatic reactions, and quantifying the decrease in GSH concentration within the reaction system by 1 μmol/L.

pour:

y: Standard OD value – blank OD value

x: concentration of the standard

a: Slope of the standard curve

b: Intercept of the standard curve

ΔA412: Non-enzymatic well OD value – enzymatic well OD value

(0.23 + V) / (0.03 + V): Dilution factor of the enzymatic reaction system

0.1/V: In the definition of enzyme activity, this refers to the enzyme activity measured in 0.1 mL of serum (or plasma), with a sample volume of V mL.

Cpr: Protein concentration of the test sample (mg prot/mL)

f: The dilution factor of the sample prior to its addition to the detection system

V: Sample volume (mL)

1.This kit is for research use only. Should it be used for clinical diagnosis or any other purpose, our company shall not be held liable for any resulting issues, nor shall it assume any legal liability.

2.Before conducting the experiment, please carefully read the instruction manual, properly calibrate the instrument, and strictly follow the instructions when performing the experiment.

3.During the experiment, please wear a lab coat and latex gloves to ensure proper personal protection.

4.The detection range of the assay kit does not correspond to the concentration range of the analyte in the sample. If the concentration of the analyte in the sample is too high or too low, please appropriately dilute or concentrate the sample.

5.If the sample being tested is not among the sample types listed in the product instructions, it is recommended to first conduct a preliminary experiment to verify the assay's validity.

6.The final experimental results are closely dependent on factors such as the efficacy of the reagents, the operator's proper procedures, and the experimental environment. Our company assumes liability solely for the assay kits themselves and is not liable for sample consumption resulting from the use of these kits. Before use, please carefully consider the potential sample volume required and reserve sufficient samples.