The mTOR (mechanistic Target of Rapamycin) ELISA (Enzyme-Linked Immunosorbent Assay) Kit is designed to measure the levels of mTOR protein or its phosphorylated forms in biological samples. mTOR is a crucial kinase involved in cell growth, proliferation, and metabolism, and its dysregulation is linked to various diseases, including cancer, metabolic disorders, and neurological diseases. The mTOR ELISA Kit allows researchers to quantify mTOR and its phosphorylated states, providing insights into cellular signaling and disease mechanisms.
Key Components and Technical Details
Principle of the Assay
- The mTOR ELISA typically uses a sandwich ELISA format, where capture antibodies specific to mTOR or its phosphorylated forms are used to bind and detect mTOR in the sample.
Kit Components
- Coated Microplate: A 96-well plate pre-coated with anti-mTOR antibodies specific to the mTOR protein or its phosphorylated forms.
- Standards: Recombinant mTOR proteins or phosphorylated mTOR forms at known concentrations used to create a standard curve.
- Detection Antibody: An antibody specific to mTOR or its phosphorylated forms, often conjugated with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
- Substrate Solution: A reagent that reacts with the enzyme linked to the detection antibody to produce a colorimetric or chemiluminescent signal proportional to the amount of mTOR.
- Stop Solution: Usually an acidic solution, it halts the enzymatic reaction and stabilizes the color for measurement.
- Washing Buffer: Used to wash away unbound substances and minimize background noise.
Sample Preparation
- Cell Lysates/Tissue Homogenates: Samples should be prepared by homogenizing and lysing tissues or cells to effectively extract mTOR proteins or phosphorylated forms.
- Serum/Plasma: For blood-based assays, proper handling and storage are crucial to maintain mTOR stability and avoid degradation.
Assay Procedure
- Coating: The microplate wells are pre-coated with antibodies specific to mTOR or its phosphorylated forms.
- Blocking: Non-specific binding sites are blocked using a blocking buffer to prevent false positives.
- Sample Incubation: Samples or standards are added to the wells to allow mTOR or phosphorylated mTOR to bind to the coated antibodies.
- Washing: Wells are washed to remove unbound substances and reduce background interference.
- Detection: The detection antibody is added, which binds specifically to mTOR or its phosphorylated forms and is conjugated with an enzyme.
- Substrate Reaction: The substrate solution is added, leading to a colorimetric or chemiluminescent reaction proportional to the amount of mTOR.
- Stop Reaction: A stop solution is added to halt the enzymatic reaction and stabilize the color.
- Reading: Optical density or fluorescence/intensity is measured using a microplate reader at the appropriate wavelength (usually 450 nm for colorimetric assays).
Quantification
- The standard curve created from known concentrations of mTOR or phosphorylated mTOR standards is used to determine the concentration of mTOR in the samples based on optical density readings.
Applications
- Cancer Research: Studying mTOR signaling pathways and their alterations in cancer.
- Metabolic Disorders: Investigating the role of mTOR in metabolic diseases such as diabetes and obesity.
- Neurodegenerative Diseases: Exploring the involvement of mTOR in neurological disorders and aging.
- Drug Development: Evaluating the effects of potential therapeutic agents targeting mTOR pathways.
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