Technical Manual for Human Glutathione Peroxidase 4 (GPX4) ELISA Detection Kit

BS-9843 Human Glutathione Peroxidase 4 (GPX4) ELISA Kit

Product Overview

This kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) for in vitro quantitative detection of glutathione peroxidase 4 (GPX4) concentration in samples such as human serum, plasma, tissue homogenate, cell lysate, and cell culture supernatant. GPX4 is a key member of the glutathione peroxidase family, which plays a central role in inhibiting iron death, maintaining cell membrane integrity, and resisting oxidative stress by catalyzing the clearance of lipid peroxides through reduced glutathione (GSH).

Detection Principle

Double antibody sandwich method: The coated antibody binds to GPX4 in the sample, and biotinylated antibody forms a complex. Horseradish peroxidase (HRP) labeled streptavidin catalyzes the colorimetric reaction, and the colorimetric intensity is proportional to the concentration of GPX4 in the sample.

Specificity: It can specifically recognize GPX4 without cross reactivity with analogs, ensuring detection accuracy.

Composition of reagent kit

Component specifications/description

Pre packaged microplate 96 well plate containing anti human GPX4 antibody

Standard freeze-dried powder, recombinant human GPX4 protein, redissolved with diluent before use

Biotin labeled antibody liquid, HRP labeled anti human GPX4 antibody

Chromogenic substrate (TMB) liquid, 3,3 ', 5,5' - tetramethylbenzidine solution

Termination solution 2M sulfuric acid solution

Wash buffer 20 x concentrated solution, containing Tween-20

Sample diluent buffer, used to dilute high concentration samples

Transparent sealing film, used for sealing microporous plates

Sample processing and preservation

Sample types: serum, plasma, tissue homogenate, cell lysate, cell culture supernatant, etc.

Handling method:

Serum/plasma: After room temperature coagulation, centrifuge (3000 rpm, 10 minutes) and collect the supernatant.

Tissue homogenate: Prepare a 10% homogenate solution with physiological saline and centrifuge to obtain the supernatant.

Cell lysate: After lysis, centrifuge (15000 rpm, 10 minutes) and take the supernatant.

Storage conditions: For short-term (≤ 7 days) storage at 2-8 ℃, for long-term (>7 days) storage, it should be packaged and frozen at -20 ℃ to avoid repeated freeze-thaw cycles.

operating steps

Standard dilution: Dilute the standard in sequence with sample diluent to 1000 pg/mL、500 pg/mL、250 pg/mL、125 pg/mL、62.5 pg/mL、31.25 pg/mL、15.63 pg/mL，0 pg/mL For blank control.

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

Washing: Discard the liquid in the well, fill each well with washing solution, let it stand for 30 seconds, discard, repeat 5 times, and pat dry.

Add biotin labeled antibody: Add 100 μ L biotin labeled antibody to each well and incubate at 37 ℃ for 60 minutes.

Add chromogenic substrate: Add 90 μ L TMB substrate to each well and incubate at 37 ℃ in the dark for 15-30 minutes.

Termination reaction: Add 50 μ L of termination solution to each well, mix well, and immediately measure the absorbance.

Result Calculation

Instrument settings: ELISA reader wavelength 450 nm (reference wavelength 630 nm).

Data processing: Plot a standard curve with standard concentration (pg/mL) as the horizontal axis and OD value as the vertical axis. The sample concentration is calculated by the curve equation and multiplied by the dilution factor.

Sensitivity: 7.69 pg/mL, detection range 15.62-1000 pg/mL.

Precautions

Cross reactivity: does not cross react with analogues such as GPX1, GPX2, GPX3, etc.

Operating standards:

Wash thoroughly to avoid residual liquids affecting the results.

The color development time should be controlled within 15-30 minutes to avoid data deviation caused by excessive color development.

Avoid repeated freezing and thawing of the sample to prevent protein degradation.

Quality control: It is recommended to set up double hole retesting for each batch of experiments, and a CV value<10% is considered qualified.

Storage conditions

Unopened reagent kit: stored at 2-8 ℃ in the dark, with a shelf life of 6 months.

After opening: Standard samples, antibodies, substrates, and other components should be packaged and stored according to the instructions to avoid repeated freezing and thawing.

This manual is only for scientific research purposes and is not applicable for clinical diagnosis. Please read the complete instructions carefully before operation and comply with laboratory safety regulations.

Note: For scientific research purposes, the above information is for reference only.

The human glutathione peroxidase 4 (GPX4) ELISA kit has always regarded quality control as the lifeblood of the enterprise and pursued continuous improvement of its competitiveness. The company always adheres to the corporate spirit of abiding by laws and regulations, being strict with oneself, being tolerant and willing to take on responsibilities as the standard in its operations. It maintains and expands the market with excellent quality and service, and maximizes the satisfaction of customer needs. Our development goal is to achieve mutual benefit with our customers.