Glutathione Peroxidase (GSH-Px) Activity Assay Kit (DTNB, Colorimetric Method) - BioReagent

Cat. No.: G1505754

GRADE & PURITY BioReagent

Synonyms

GPX Activity Assay Kit

Storage

Store at 2-8°C,Protected from light,Room temperature,Store at -20°C

Shipped In

Ice chest + Ice pads

Application

Cell Metabolism, Enzyme activity assay

Overview

  Glutathione Peroxidase (GSH-Px) is a selenium-containing, water-soluble tetrameric proteolytic enzyme distributed in almost all tissues. Its activity can change significantly under certain pathological conditions. This enzyme scavenges peroxides within living cells and plays a key role in protecting cells from free radical damage. Intracellular lipids are prone to react with free radicals, producing lipid peroxides. GSH-Px not only eliminates free radicals and their derivatives but also, together with Catalase (CAT), Phospholipid Hydroperoxide Glutathione Peroxidase (PH-GSH-Px), and Glutathione S-Transferase (GST), forms a multi-level system for reducing organic hydroperoxides with different substrate specificities. This system reduces the formation of lipid peroxides and enhances the body's capacity to resist oxidative damage.

Assay Principle

  GSH-Px catalyzes the reaction between hydrogen peroxide (H₂O₂) and reduced glutathione (GSH) to produce H₂O and oxidized glutathione (GSSG). The activity of GSH-Px can be represented by the rate of this enzymatic reaction. By measuring the consumption of reduced glutathione in this reaction, the enzyme activity can be determined. The activity of GSH-Px is expressed as the rate of GSH reaction. Since these two substrates can also undergo redox reactions without the enzyme (non-enzymatic reaction), the final calculation of enzyme activity must subtract the portion of GSH reduction caused by the non-enzymatic reaction. The GSH content is measured based on the reaction between GSH and 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), which produces a stable yellow 5-thio-2-nitrobenzoic acid anion. The change in absorbance at 412 nm is used to determine Glutathione Peroxidase activity.

G1505754	Component	50T	Storage
G1505754A	Sample Homogenization Buffer	50 mL	RT
G1505754B	GSH	15.4 mg	2-8℃
G1505754C	GSH Preparation Solution	10 mL	RT
G1505754D	Oxidizing Agent	1 mL×2	2-8℃. Store in the dark.
G1505754E	Acidic Precipitation Agent	100 mL	RT
G1505754F	GSH-Px Assay Buffer	62.5 mL	RT
G1505754G	Benzoic Acid Chromogenic Solution	15 mL	-20℃. Store in the dark.

Required Materials Not Provided

1. Physiological saline or PBS

2. Centrifuge tubes or EP tubes, Spectrophotometer, Cuvettes, Water bath or incubator, Centrifuge

Experimental Procedure

1. Sample Preparation

1.1 Serum/Plasma Samples

• Serum or plasma separated from the sample should not be hemolyzed. If present, remove red blood cells before assay. If exceeding the detection range, dilute with physiological saline before assay.

• Simple method for RBC removal from serum: Collect blood in an anticoagulant tube, mix by inverting. Take at least 500 µL whole blood, centrifuge at 3000 rpm/min for 5 min at 4°C, discard supernatant. Resuspend the RBC pellet in 10 volumes of pre-chilled Sample Homogenization Buffer, centrifuge again (3000 rpm/min, 5 min, 4°C), discard supernatant. Add approx. 4 volumes of pre-chilled ddH₂O to lyse the RBC pellet, centrifuge at 12,000 rpm/min for 5 min, collect supernatant. Alternatively, use products like ACK Lysis Buffer to remove RBCs, then collect supernatant.

1.2 Tissue Samples

• Perfuse animals with physiological saline containing 20 U/ml Heparin to remove blood before collecting tissue samples.

• Homogenize tissue on ice or at 4°C using a glass homogenizer with 200 µL Sample Homogenization Buffer per 20 mg tissue.

• Centrifuge at 12,000 rpm/min for 10 min at 4°C. Collect the supernatant.

1.3 Cell Samples

• For adherent cells: Avoid trypsin digestion as it may affect subsequent enzyme activity assays. Use a cell scraper or EDTA to harvest cells. Wash cells once with PBS or physiological saline.

• Homogenize cells on ice or at 4°C using a glass homogenizer with 300-500 µL Homogenization Buffer per 10⁶ cells.

• Centrifuge at 12,000 rpm/min for 10 min at 4°C. Collect the supernatant for enzyme assay.

• Alternatively: Lyse cells using RIPA Lysis Buffer according to the manufacturer's instructions, using 100-200 µL lysis buffer per 10⁶ cells. Collect the supernatant.

1.4 Plant Samples

• Weigh 0.2 g of fresh or -80°C frozen sample, place in a pre-chilled mortar.

• Add 2 mL of pre-chilled Phosphate Buffer (0.05 M, pH 7.0).

• Grind or homogenize on ice.

• Transfer to a centrifuge tube, centrifuge at 12,000 rpm/min for 10-15 min at 4°C.

• Collect the supernatant for enzyme assay.

2. Preparation of GSH Working Solution

• Add 0.5 mL ddH₂O to the 15.4 mg GSH vial. Dissolve and mix thoroughly to obtain the GSH Stock Solution (100 mmol/L).

• Aliquot immediately and store at -20°C.

• For the GSH Working Solution (1 mmol/L), mix GSH Preparation Solution and GSH Stock Solution (100 mmol/L) at a 99:1 ratio.

• This working solution can be stored at 4°C for 1 day after preparation.

3. Preparation of Oxidizing Working Solution

• Accurately pipette 0.1 mL of the Oxidizing Agent into 6.5 mL ddH₂O to prepare the Oxidizing Stock Solution (100X). Store at 4°C.

• Before use, accurately pipette 0.1 mL of the Oxidizing Stock Solution (100X) into 9.9 mL ddH₂O to prepare the Oxidizing Working Solution.

• Store at 4°C, stable for 1 day.

4. GSH-Px Enzymatic Reaction

• Set up Blank Control, Background Control, and Test tubes in centrifuge tubes according to the table below.

• Add reagents in the specified order:

Reagent (mL)	Blank Control Tube	Background Control Tube	Test Tube
GSH Working Solution (1 mmol/L)	—	0.2	0.2
Sample	—	—	0.2
ddH₂O	0.2	0.2	—

Mix well and incubate at 37°C for 5 min.

Oxidizing Working Solution (pre-warmed 37°C)

—

0.1

0.1

Mix well and incubate at 37°C for 5 min.

Acidic Precipitation Agent

0.8

2.0

2.0

Centrifuge at 3500 g for 10 min.

Supernatant Collected

—

1.0

1.0

5. GSH-Px Chromogenic Reaction

• Set up Blank Control, Background Control, and Test wells/tubes in a 96-well plate or centrifuge tubes according to the table below.

• Add reagents in the specified order:

Reagent (mL)	Blank Control Tube	Background Control Tube	Test Tube
Supernatant from Step 4	—	1.0	1.0
Blank Control from Step 4	1.0	—	—
GSH-Px Assay Buffer	1.25	1.25	1.25
Benzoic Acid Chromogenic Solution	0.25	0.25	0.25

6. GSH-Px Measurement

Mix well and incubate at room temperature for 1 minute. 

Zero the instrument with ddH₂O. 

 Measure the absorbance at 412 nm using a spectrophotometer or microplate reader (recorded as A_Blank, A_Background, A_Test). 

 Note for spectrophotometers: Use a 1 cm light path cuvette; adjust volumes based on the cuvette's minimum requirement. 

 Note for microplate readers: Add 250 µL per well if using a 96-well plate. 

 Typically, A_Blank ranges from 0.003 to 0.05, and A_Background ranges from 0.1 to 0.3. 

7. Calculation 

Definition of GSH-Px Activity Unit: Excluding non-enzymatic reactions, one unit of GSH-Px activity is defined as the amount of enzyme required to catalyze the oxidation (consumption) of 1 μmol of GSH per minute per liter of serum at 37°C. 

GSH-Px (U/L) = (A_Background - A_Test) / (A_Background - A_Blank) × 200 Parameter Definitions: 

 A_Blank: Absorbance of the Blank Control 

 A_Background: Absorbance of the Background Control 

 A_Test: Absorbance of the Test sample 

 200: Derived from 1000 (mL) / 5 (min) – relates unit definition to assay conditions. 

 Notes: 

① The unit for [Glutathione Peroxidase activity in the detection system] is U/L, which is equivalent to mU/mL. 

② [Glutathione Peroxidase activity in the sample (e.g., tissue)] = [GSH-Px activity in detection system] × [Dilution Factor] / [Sample Protein Concentration] 

* Unit for [GSH-Px activity in sample]: U/mg or mU/mg protein 

* Unit for [Sample Protein Concentration]: mg/mL ③ Calculation Example: 

* Sample protein concentration: 0.5 mg/mL, assayed after a 2-fold dilution. 

* Typical values: A_Blank ~0.003-0.05, A_Background ~0.1-0.3. 

* If A_Background = 0.30, A_Test = 0.20, A_Blank = 0.003: 

* Sample GSH-Px activity = (0.30 - 0.20) / (0.30 - 0.003) × 200 = 67.34 U/L 

* Sample GSH-Px activity = 67.34 U/L × 2 / (0.5 mg/mL) = 269.36 mU/mg protein 

Reference Interval: Adult Serum GSH-Px: 115 ~ 140 U/L

Precautions

1. Avoid repeated freeze-thaw cycles for the low-temperature reagents to prevent inactivation or reduced efficiency.

2. All oxidizing or reducing agents can interfere with this assay. If unavoidable in the sample (e.g., DTT, β-mercaptoethanol), keep the total concentration of such reductants below 0.1 mM. Note: 0.15 mM DTT can inhibit 40% of enzyme activity.

3. Common detergents like Triton X-100 and Tween 20 often contain high levels of peroxides, which can interfere. If necessary, use high-purity grades specified to have low peroxide content.

4. Assay samples immediately after preparation, or store at -80°C for later analysis.

5. Strictly control the reaction temperature to minimize errors.

6. Use reagents promptly after opening to avoid affecting subsequent experimental results.

7. For your safety and health, wear lab coats and disposable gloves during operation.

Specifications

Synonyms

GPX Activity Assay Kit

Specifications & Purity

BioReagent

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Storage

Store at 2-8°C, Protected from light, Room temperature, Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive