Body fluid reduction glutathione concentration colorimetric quantitative detection kit instruction manual

Body fluid reduction glutathione concentration colorimetric quantitative detection kit product manual (Chinese version)

The main purpose

REDUCED GLUTATHIONE concentration colorimetric quantitative detection reagent is a product designed to react with reduced glutathione using Ellman reagent to produce yellow 5-mercapto-2-nitrobenzoic acid product. The change in the peak absorbance is an authoritative and classical technique using colorimetry to determine the reduced glutathione content in a sample. The technology has been carefully developed and successfully tested. It is suitable for the detection of reduced glutathione in various body fluids including urine, cerebrospinal fluid, saliva, semen and the like. The product is strictly sterile, ready to use, simple in operation, stable in performance and good in repeatability.

technical background

Glutathione is a tripeptide molecule, γ-glutamylcysteinylglycine. It is also the most abundant compound containing Thiol. It is widely found in animals and plants. In bacteria and yeast cells. And 90 to 95% exist in a reduced form. Its function is 1) as a nucleophilic substrate of glutathione transferase, detoxification of exogenous chemicals (2enobiotics); 2) as an electron donor of glutathione peroxidase, Hydroperoxides are reduced; 3) participate in the maintenance of Amino Acid transport and protein sulfhydryls, as well as antioxidant effects. The level of intracellular glutathione is a sensitive indicator of cell health. Reaction of the thiol group of reduced glutathione with Ellman's reagent 5,5-dithiobis-(2-nitrobenzoic acid)[DTNB] After that, a yellow 5-thio-2-nitrobenzoic acid (TNB) was produced, and the content of reduced glutathione was quantitatively analyzed by the change in absorption peak (wavelength at 412 nm). , the way of reaction is:

product content

Anti-interference solution (Reagent A) ml

Buffer (Reagent B) ml

Substrate solution (Reagent C) ml

Standard solution (Reagent D) microliter

Diluent (Reagent E) ml

Product manual 1 copy

storage method

Store anti-interference liquid (Reagent A) and buffer (Reagent B) in a refrigerator at 4 °C, and store the rest in a refrigerator at -20 °C; Reagent C and Reagent D avoid light; June

User-supplied

1.5 ml centrifuge tube: container for sample and standard preparation

Micro Tabletop Centrifuge: for sample processing

Constant temperature sink: for reaction incubation

96-well plate or 200 μl 1 cm diameter cuvette: container for colorimetry

Microplate reader or spectrophotometer: for sample colorimetric analysis

Experimental procedure

Before the start of the experiment, set the spectrophotometer (temperature is 25 ° C): the wavelength is 412 nm, and set to zero. Then do the following.

First, sample preparation ( 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
• Immediately placed in an ice bath or placed in a -70 ° C freezer
• (Selecting step) Pipette 10 [mu] l for protein quantification (Note: We recommend a protein concentration using the Bradford Assay Kit -GMS30030.1)
• Pipette 500 μl into a new 1.5 ml centrifuge tube
• Add pre-cooled xx microliters of de-interference solution ( Reagent A )
• Vortex for 5 seconds, mix thoroughly
• Place in a 4°C benchtop centrifuge for 10 minutes at a speed of 10,000g
• Carefully remove the supernatant to another new pre-cooled 1.5 ml centrifuge tube
• Store in a refrigerator at -70 ° C or in an ice bath (recommended within 2 hours)

Second, standard sample preparation

• Prepare 5 1.5 ml tubes, labeled 1 to 5
• Add xx microliters of diluent ( Reagent E ) to tubes 2 through 5, respectively.
• Pipette xx microliters of standard solution ( Reagent D ) to tube 1
• Carefully remove the standard solution ( Reagent D ) of the xx microliter No. 1 tube into the No. 2 tube and mix well.
• Carefully remove the xx microliter No. 2 tube diluted standard solution ( Reagent D ) into the No. 3 tube and mix.
• Carefully remove xx μl of No. 3 tube diluted standard solution ( Reagent D ) into tube No. 4 and mix well.
• Put the 1 to 5 tubes into the ice tank for use, avoiding the light; the standard tube concentration is shown in the table below.

• Standard curve determination

• Transfer xx μl of buffer ( Reagent B ) to the new cuvette
• Add 20 μl of the above prepared standard solution
• Pour up and down several times and mix
• Incubate for 5 minutes at 37 ° C
• Add xx microliters of substrate solution ( Reagent C )
• Pour up and down several times and mix
• Incubate for 10 minutes at room temperature
• Immediately put into the spectrophotometer to detect: obtain absorbance readings
• Repeat the experiment steps 1 to 10 four times
• Construct a standard curve: the ordinate (Y-axis) is the absorbance reading; the abscissa (X-axis) is the standard reduced glutathione concentration (micromol/L)

• Sample determination

• Transfer xx μl of buffer ( Reagent B ) to the new cuvette
• Add 20 μl of the sample to be tested prepared above
• Pour up and down several times and mix
• Incubate for 5 minutes at 37 ° C
• Add xx microliters of substrate solution ( Reagent C )
• Pour up and down several times and mix
• Incubate for 10 minutes at room temperature
• Immediately put into the spectrophotometer to detect: obtain absorbance readings
• The sample corresponding to the reduced glutathione concentration (micromol/L) was obtained according to the standard curve.
• Calculate the actual reduced glutathione concentration of the sample (micromoles / liter)

• Then determined

• Mark the 96-well plate: standard sample and sample to be tested
• Transfer xx μl of buffer ( Reagent B ) to the corresponding well
• Add 20 μl of the above prepared standard solution or sample to be tested
• Gently shake the 96-well plate
• Incubate for 5 minutes at 37 ° C
• Add xx microliters of substrate solution ( Reagent C )
• Gently shake the 96-well plate
• Incubate for 10 minutes at room temperature
• Immediately put into the microplate reader test: obtain absorbance readings
• Construct a standard curve: the ordinate (Y-axis) is the absorbance reading; the abscissa (X-axis) is the standard reduced glutathione concentration (micromol/L)
• The sample corresponding to the reduced glutathione concentration (micromol/L) was obtained according to the standard curve.
• Calculate the actual reduced glutathione concentration of the sample (micromoles / liter)

Precautions

• This product is 50 operations, including standard samples
• Wear gloves when handling
• This product detects 1 to 100 micromoles of reduced glutathione
• If the interference sample liquid (Reagent A) process and should be completed within 2 hours detected; if the sample needs to be saved later retested, without interference recommended solution (Reagent A) processing, stored directly at -70 ℃ in
• Avoid all kinds of reducing chemicals in the sample, including 2-mercaptoethanol, dithiothreitol (DTT), cysteine ​​(cysteine) and N-ethyl maleimide. (N-Ethylmaleimide; NEM), etc.
• The range of sample reduced glutathione concentration is unclear. It is recommended to serially dilute the sample 2 to 3 times.
• If the user does not have a 412nm wavelength filter, you can use the 405nm wavelength instead.
• After the colorimetric determination, the cuvette must be thoroughly cleaned.
• If the concentration of the sample to be tested is too high, the sample can be diluted.
• If the concentration of the sample to be tested is too low, concentrate the sample or extend the incubation time to 30 or 40 minutes.
• The company provides a series of glutathione analysis reagent products

Quality Standard

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

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