Hypoxia-inducible factor-1α (HIF-1α) Assay Method: Introduction
Objective: This kit is designed to quantify the levels of hypoxia-inducible factor-1α (HIF-1α) in rat serum, plasma, and other biological fluids. It provides a reliable and accurate method for measuring HIF-1α concentrations, which is essential in studying hypoxic conditions and related physiological or pathological processes.
Principle of the Experiment: The assay is based on the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique. A microplate is pre-coated with a specific monoclonal antibody against rat HIF-1α. After incubation with the sample, HIF-1α present in the specimen binds to the immobilized antibody. Subsequently, a horseradish peroxidase (HRP)-labeled secondary antibody is added, forming a complex of antibody-HIF-1α-enzyme-labeled antibody. Following a thorough wash step to remove unbound components, the substrate TMB (3,3',5,5'-tetramethylbenzidine) is introduced. Under the catalytic action of HRP, TMB turns blue and then changes to yellow upon addition of an acidic stop solution. The intensity of the color is directly proportional to the concentration of HIF-1α in the sample.
The optical density (OD) at 450 nm is measured using a microplate reader. By comparing the OD values of the test samples with a standard curve generated from known concentrations of HIF-1α, the exact level of HIF-1α in each sample can be determined accurately. This method ensures high specificity and sensitivity, making it ideal for research and diagnostic applications.
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