Body fluid xanthine oxidase activity colorimetric quantitative detection kit instructions

Humoral activity of xanthine oxidase colorimetric quantitative detection kit product specification (Chinese)

The main purpose

YIJI body fluid xanthine oxidase activity colorimetric quantitative detection reagent is a kind of hydrogen peroxide generated by coupling reaction system of xanthine oxidase and peroxidase enzyme, and reacts with chromogenic dye to produce quinone The change in the absorbance peak exhibited by the product is an authoritative and classical technique for determining enzyme activity in body fluid samples using colorimetry. The technology has been carefully developed and successfully tested. It is suitable for the detection of xanthine oxidase activity and screening of inhibitors in various animal or human body fluid samples (blood, semen, cerebrospinal fluid, urine, etc.). The product is strictly sterile, ready to use, simple in operation and stable in performance.

technical background

Xanthine oxidase (XO; EC1.17.3.2), also known as xanthine oxidoreductase (xanthine oxidoreductase) is a complex molybdoflavoenzyme (mlybdoflavoenzyme), which is the terminal of sputum catabolism in human body. Enzymes are found in all living organisms. In mammals, it is mainly expressed in the liver and small intestine. Xanthine oxidase is a macromolecular structure composed of single molecules and multiple elements. It has a molecular weight of 270KD, contains two flavin molecules, two molybdenum atoms, and eight iron atoms, and participates in the electron transport of the respiratory chain. Xanthine oxidase catalyzes the hydroxylation of hypoxanthine, producing xanthine, which is then oxidized to uric acid. In addition, xanthine oxidase can act as a NADH oxidase, producing superoxide anion and hydrogen peroxide, affecting various cellular molecular components, leading to cell degradation and death, as well as cardiovascular disease. The activity of xanthine oxidase in liver tissue of patients with liver disease is 1000 times higher than that of normal people. Xanthine oxidase deficiency will cause xanthinuria, leading to kidney failure. Based on the substrate hypoxanthine, hydrogen peroxide is generated by the oxidation of xanthine oxidase, and then with the action of peroxidase, p- hydroxybenzoic acid It reacts with 4-aminoantipyrine to form a quinoneimine, and the activity of xanthine oxidase is quantitatively analyzed by the change in its absorption peak (wavelength at 510 nm). The reaction system is:

product content

YIJI buffer (Reagent A) ml

YIJI substrate solution (Reagent B) ml

YIJI Reaction Solution (Reagent C) ML

YIJI negative solution (Reagent D) microliter

Product manual 1 copy

storage method

Stored in a -20 ° C refrigerator; YIJI substrate solution (Reagent B) to avoid light; effective guarantee for June

User-supplied

Anticoagulant: a container for blood collection

1.5 ml centrifuge tube: container for sample handling and storage

4°C (micro) benchtop centrifuge: for sample processing

Incubator: for reaction incubation

Cuvette or plate: a container for colorimetry

Spectrophotometer or microplate reader: for colorimetric analysis

Experimental procedure

  • Sample preparation

Option 1: plasma sample

  • Prepare heparin or ACD anticoagulation tube (Note: Avoid using EDTA anticoagulation tube)
  • Take 1 ml of blood and place it in an anticoagulation tube
  • Place in a 4°C mini tabletop centrifuge for 10 minutes at a speed of 700g (or 3000RPM, eg eppendorf 5415)
  • Carefully remove the upper yellow liquid into a new 1.5 ml centrifuge tube - this is the plasma component (note: avoid touching the white liquid layer )
  • Pipette 10 [mu] l for protein quantification (Note: the concentration recommended YIJI Bradford protein quantification kit - YJ 30030.1)
  • Immediately placed in an ice bath or placed in a -70 ° C freezer

Option 2: serum samples

1. Prepare a storage tube without anticoagulant

  • Take 1 ml of blood and place it in the storage tube
  • Allow to stand for 30 minutes at room temperature until blood clots
  • Place in a 4°C mini tabletop centrifuge for 15 minutes at a speed of 2000g (or 5000RPM, eg eppendorf 5415)
  • Carefully remove the upper yellow liquid into a new 1.5 ml centrifuge tube - this is the serum component (Note: Avoid touching the white liquid layer )
  • Pipette 10 [mu] l for protein quantification (Note: the concentration recommended YIJI Bradford protein quantification kit - YJ 30030.1)
  • Immediately placed in an ice bath or placed in a -70 ° C freezer

Option 3: urine / cerebrospinal fluid / saliva / semen samples

  • Prepare the 1.5 ml centrifuge tube
  • Pipette 1 ml of liquid into the centrifuge tube
  • Place in a 4°C mini tabletop centrifuge for 10 minutes at a speed of 700g (or 3000RPM, eg eppendorf 5415)
  • Carefully remove the supernatant into a new 1.5 ml centrifuge tube
  • Pipette 10 [mu] l for protein quantification (Note: the concentration recommended YIJI Bradford protein quantification kit - YJ 30030.1)
  • Immediately placed in an ice bath or placed in a -70 ° C freezer

  • Preparation for measurement

  • Turn on and set the spectrophotometer (temperature is 37 ° C): wavelength 510nm, interval 5 minutes, reading 3 times (15 minutes total), and set zero
  • Was removed from the freezer -20 ℃ agent, placed in an ice melted trough; YIJI substrate solution (Reagent B) protected from light; YIJI at room temperature equilibrium buffer (Reagent A)

  • Background control

  • Transfer xx μl of YIJI Buffer ( Reagent A ) to a new cuvette
  • Add xx microliters of YIJI substrate solution ( Reagent B )
  • Add xx microliters of YIJI reaction solution ( Reagent C )
  • Pour up and down several times and mix
  • Incubate for 3 minutes at 37 ° C
  • Add xx microliters of YIJI negative solution ( Reagent D )
  • Pour up and down several times, mix (limited to 3 seconds)
  • Immediately put into the spectrophotometer test, this is the background air control (15 minutes reading - 0 minutes reading)

  • Sample determination

  • Transfer xx μl of YIJI Buffer ( Reagent A ) to a new cuvette
  • Add xx microliters of YIJI substrate solution ( Reagent B )
  • Add xx microliters of YIJI reaction solution ( Reagent C )
  • Pour up and down several times and mix
  • Incubate for 3 minutes at 37 ° C
  • Add 50 μl of the sample to be tested (50 μg of protein) (Note: the sample should be clear )
  • Pour up and down several times, mix (limited to 3 seconds)
  • Immediately put into the spectrophotometer test, this is the sample reading (15 minutes reading - 0 minutes reading)

Fifth, calculate the sample activity

  • Enzyme plate assay

  • Mark the 96-well microtiter plate: background control and sample
  • Transfer xx μl of YIJI buffer ( Reagent A ) to a 96-well plate
  • Add xx microliters of YIJI substrate solution ( Reagent B )
  • Add xx microliters of YIJI reaction solution ( Reagent C )
  • Gently shake the 96-well microtiter plate
  • Incubate for 3 minutes at 37 ° C
  • Add xx μl of YIJI negative solution ( Reagent D ) or sample to be tested (50 μg protein) to the appropriate wells (note: the sample should be clear )
  • Gently shake the plate
  • Immediately put into the microplate reader test: 0 minute reading and 15 minute reading
  • Activity calculation:

Precautions

  • This product is 20 operations, including background control
  • Wear gloves when handling
  • Only 1 time for background determination during system operation
  • Samples must be clarified, it is vital
  • Colorimetric determination within 3 seconds after loading
  • The measured value changes from low to high; the measurement lasts for 15 minutes.
  • After the colorimetric determination, the cuvette must be thoroughly cleaned.
  • The sample is measured for 15 minutes and the reading is higher than 0 minutes.
  • It is recommended that the sample protein concentration to be tested be 50 μg/50 μl (the company provides YIJI Bradford Protein Concentration Quantitation Kit - YJ30030.1)
  • If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
  • The unit activity of xanthine oxidase is defined as the amount of enzyme required to convert 1 micromolar xanthine to uric acid per minute at 37 ° C, pH 7.5 as an active unit.
  • The company provides a series of oxidase detection reagent products

Quality Standard

  • This product has been certified to be stable.
  • This product has been identified and sensitive

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