Glutathione Peroxidase Activity Colorimetric Quantification Test Kit Instruction Manual

Glutathione Peroxidase Activity Colorimetric Quantification Test Kit Product Manual (Chinese version)

The main purpose

Glutathione peroxidase (GSH PX) activity colorimetric quantitative detection reagent is a reduced-type smoke in an enzyme coupling reaction system designed to pass glutathione peroxidase and glutathione reductase A decrease in the peak absorbance after oxidation of the adenine dinucleotide phosphate (NADPH), an authoritative and classical technique for determining enzyme activity in a sample using colorimetry. The technology was carefully developed by master scientists and successfully demonstrated by experiments. It is suitable for the detection of total activity of glutathione peroxidase in various cells, tissues, blood, body fluids and the like. The product is strictly sterile, ready to use, simple in operation and stable in performance.

technical background

Glutathione peroxidase (GSH PX or GPX; EC 1.11.1.9) is a member of the selenoprotein family and is expressed in cytoplasmic and mitochondrial components. Its function is to catalyze various Reduction of oxides, including hydroperoxides, hydrogen peroxide, etc., protect cells from oxidative damage. The decrease in enzyme activity is associated with diseases such as Parkinson's disease and chronic glomerulonephritis. In mammals, there are four types of glutathione peroxidase: type I is a classic intracellular type; type II is a gastrointestinal type; type III is a plasma type; type IV is a phospholipid hydroperoxidation metabolism Pholipid hydroperoxide metabolizing. In addition to type IV (which is a monomer), the structure of glutathione peroxidase is a homotetramer, each monomer is 22 to 23 kd, and its active domain contains selenocysteine. Glutathione-peroxidase-based reduction of organic peroxide (ROOH), while reducing glutathione (GSH) to oxidized glutathione (GSSG); Under the action of glutathione reductase (GR), oxidized glutathione is reduced to reduced glutathione, while reduced nicotinamide adenine dinucleotide Phosphate; NADPH) is converted to oxidized nicotinamide adenine dinucleotide phosphate (NADP) to quantify glutathione peroxide based on its reduced absorbance peak after oxidation (340 nm wavelength) The total activity of the enzyme. The glutathione peroxidase coupling cycle reaction system is:

Glutathione peroxidase

ROOH + 2GSH → ROOH + GSSG + H2O

Glutathione reductase

GSSG + NADPH → 2 GSH + NADP +

(high absorption peak) (low absorption peak)

product content

Buffer (Reagent A) 5 ml

Background solution (Reagent B) 500 μl

Reaction solution (Reagent C) 500 μl

Negative Liquid (Reagent D) 500 μl

Product manual 1 copy

storage method

Store in a -20 ° C refrigerator; Reagent B and Reagent C to avoid light; effective June

User-supplied

1.5 ml centrifuge tube: container for reaction solution preparation

Cuvette: a container for colorimetry

Spectrophotometer: for colorimetric analysis

Microplate reader: for colorimetric analysis of trace samples

Experimental procedure

  • Preparation for measurement

  • Turn on and set the spectrophotometer (temperature is 25 ° C): wavelength 340nm, interval 60 seconds, reading 6 times (5 minutes total), and set zero
  • Reagents removed from -20 ℃ refrigerator, an ice melting trough; background solution (Reagent B) protected from light

  • Background control

  • Transfer 190 μl of buffer ( Reagent A ) to the new cuvette
  • Add 10 μl of negative solution ( Reagent D )
  • Pour up and down several times and mix
  • Incubate for 2 minutes at 25 ° C
  • (selection step) put into the spectrophotometer, set to zero
  • (selection step) remove the cuvette
  • Add 25 μl of the base solution ( Reagent B )
  • Add 25 μl of reaction solution ( Reagent C )
  • Pour up and down several times, mix (limited to 3 seconds)
  • Immediately put into the spectrophotometer test, this is the background air control (0 minutes reading - 5 minutes reading)

  • Sample determination

  • Transfer 190 μl of buffer ( Reagent A ) to the new cuvette
  • Add 10 μl of the sample to be tested (20 μg of protein) (Note: the sample should be clear )
  • Pour up and down several times and mix
  • Incubate for 2 minutes at 25 ° C
  • (selection step) put into the spectrophotometer, set to zero
  • (selection step) remove the cuvette
  • Add 25 μl of the base solution ( Reagent B )
  • Add 25 μl of reaction solution ( Reagent C )
  • Pour up and down several times, mix (limited to 3 seconds)
  • Immediately put into the spectrophotometer test, this is the sample reading (0 minutes reading - 5 minutes reading)

Fourth, calculate sample activity

[(Sample reading - background reading) X sample dilution factor X 0.25 (system capacity; ml)] ÷ [0.01 (sample capacity; ml) X 6.22 (mmol absorbance) X 5 (minutes)] = unit / ml ÷ ( Sample protein concentration) mg / ml = unit / mg

Unit = micromolar NADPH / minute

  • Enzyme plate assay

  • Mark the 96-well microtiter plate: background control and sample
  • Transfer 190 μl of buffer ( Reagent A ) to a 96-well plate.
  • Add 10 μl of negative solution ( Reagent D ) or sample to be tested (20 μg of protein) to the appropriate wells (note: the sample should be clear )
  • Gently shake the 96-well microtiter plate
  • Incubate for 2 minutes at 25 ° C
  • Add 25 μl of the base solution ( Reagent B )
  • Add 25 μl of reaction solution ( Reagent C )
  • Gently shake the plate
  • Immediately put into the microplate reader test: 0 minute reading and 5 minute reading
  • Activity calculation:

[(Sample reading - background reading) X sample dilution factor X 0.25 (system capacity; ml)] ÷ [0.01 (sample capacity; ml) X 6.22 (mmol absorbance) X 0.6 (cm) X 5 (minutes)] = Unit / ml ÷ (sample protein concentration) mg / ml = unit / mg

Unit = micromolar NADPH / minute

Precautions

  • This product is 20 operations, including background control
  • Wear gloves when handling
  • Only 1 time for background determination during system operation
  • Detection sensitivity range is 5 to 30 milliunits per milliliter
  • The sample to be tested should be free of blood, reducing agents (DTT and mercaptoethanol) and certain detergents (TWEEN-20, TRITON X-100), etc.
  • It is recommended to use cuvette determination
  • Samples must be clarified, it is vital
  • Colorimetric determination within 3 seconds after loading
  • Background empty control 0 second reading is usually 0.2 to 0.8 is ideal
  • The measured value changes from high to low; the measurement lasts for 5 minutes.
  • After the colorimetric determination, the cuvette must be thoroughly cleaned.
  • Sample measurement 0 minute reading is higher than 5 minutes reading indicates enzyme activity
  • It is recommended that the sample protein concentration to be tested be 20 μg/10 μl; if the sample enzyme activity is too low or too high, the sample size can be increased or decreased (the company provides the Bradford Protein Concentration Quantitation Kit - GMS30030.1)
  • If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
  • The glutathione peroxidase unit activity is defined as the amount of enzyme required to convert 1 micromole of reduced nicotinamide adenine dinucleotide phosphate per minute as an active unit at 25 ° C, pH 7.5.
  • The company provides a series of glutathione peroxidase detection reagent products

Quality Standard

  • This product has been certified to be stable.

This product has been identified and sensitive

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