Experimental Manual for Rat Glutathione Reductase (GSR) ELISA Kit

BS-3507 Rat Glutathione Reductase (GSR) ELISA Kit

1、 Experimental principle

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the activity of glutathione reductase (GSR) in rats. Coat the anti rat GSR antibody in a microplate, and after adding the test sample, the GSR in the sample binds to the coated antibody. Subsequently, biotinylated detection antibodies are added to form a "antibody antigen enzyme labeled antibody" complex. After adding horseradish peroxidase (HRP) labeled streptavidin (SABC), the absorbance (OD value) was measured at 450nm wavelength by TMB substrate staining, and the OD value was positively correlated with the concentration of GSR in the sample.

2、 Composition of reagent kit

Component 48 hole configuration, 96 hole configuration, storage conditions

Pre packaged enzyme-linked immunosorbent assay plates with 8 × 6 strips and 8 × 12 strips, 2-8 ℃

Standard product (freeze-dried) 1 tube 1 tube 2-8 ℃

Standard/sample diluent 10mL 15mL 2-8 ℃

Biotinylation detection antibody 60 μ L (100 ×) 120 μ L (100 ×) -20 ℃

Biotinylated antibody diluent 6mL 12mL 2-8 ℃

SABC composite 6mL 12mL 2-8 ℃

TMB color solution (A/B) 3mL each, 6mL each, avoid light 2-8 ℃

Termination solution 6mL 12mL 2-8 ℃

20 x Concentrated Wash Solution 30mL 60mL Room Temperature

2 sheets of sealing film and 4 sheets at room temperature

1 copy of instruction manual, 1 copy-

3、 Sample processing

Serum/plasma samples:

Let the whole blood stand at room temperature for 2 hours or overnight at 4 ℃, centrifuge at 1000g for 20 minutes, and collect the supernatant.

Avoid hemolysis or high blood lipid samples, which can be stored for 48 hours at 2-8 ℃; Long term storage requires -20 ℃ or -80 ℃ to avoid repeated freezing and thawing.

Organizational/cellular samples:

After homogenizing or cell lysis, centrifuge to obtain the supernatant and adjust the protein concentration to the recommended range.

4、 Experimental steps

Preparation of detergent:

Dilute 20 times the concentrated washing solution with distilled water (e.g. 1mL concentrated solution+19mL distilled water).

Standard dilution:

Take 7 centrifuge tubes and label them as 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and blank.

Add 200 μ L of standard/sample diluent to each tube, and add 200 μ L of standard stock solution to the first tube. Mix well and dilute step by step.

Sample addition and incubation:

Add 100 μ L of standard or sample to each well and incubate at 37 ℃ for 90 minutes.

Discard the liquid and wash 3 times (soak for 1-2 minutes each time).

Enzyme linked antibody reaction:

Add 100 μ L of biotinylated detection antibody (diluted 1:100) to each well and incubate at 37 ℃ for 60 minutes.

Wash three times.

SABC complex reaction:

Add 100 μ L SABC complex to each well and incubate at 37 ℃ for 30 minutes.

Wash three times.

Color rendering and termination:

Add 90 μ L of TMB chromogenic solution (A/B solution mixed equally) to each well and incubate at 37 ℃ in the dark for 15 minutes.

Add 50 μ L of termination solution to each well, mix well and measure immediately.

5、 Result calculation

Draw a standard curve with standard concentration as the x-axis (X) and OD value as the y-axis (Y).

Calculate the GSR concentration by substituting the sample OD value into the standard curve equation.

6、 Precautions

Reagent storage:

Standard products and biotinylated antibodies should be stored at -20 ℃ to avoid repeated freezing and thawing; Other components should be stored at 2-8 ℃.

Sample processing:

Avoid using samples with hemolysis, hyperlipidemia, or turbidity; The organizational sample needs to be thoroughly homogenized.

Operating standards:

Accurate pipetting is necessary to avoid cross contamination; Washing needs, residual liquid affects the results.

Instrument calibration:

The wavelength of the enzyme-linked immunosorbent assay (ELISA) reader needs to be calibrated to 450nm and the blank well needs to be zeroed.

Security protection:

Terminate the liquid containing sulfuric acid, wear gloves and goggles during operation.

7、 Expected results

Sensitivity: Can detect GSR as low as 0.156ng/mL.

Linear range: 0.156-10ng/mL.

8、 Application scope

This reagent kit is suitable for quantitative detection of GSR in rat serum, plasma, tissue homogenate, and cell culture supernatant, for scientific research purposes.

Tip: Please refer to the actual reagent kit instructions for specific operations. Before the experiment, it is necessary to read the complete instructions and confirm the integrity of the components.

Note: The above is for reference only. This reagent is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.