Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate
Trascrizione
For quantitative detection of a specific protein in mouse brain lysate using the electrochemiluminescence, ECL, immunoassay, begin with a multi-well assay plate.
The wells comprise conducting working and counter electrodes, completing the electrical circuit. A dielectric layer separates the electrodes, improving the assay sensitivity.
Add primary mouse monoclonal antibodies against the target protein to the wells. The working electrodes, with greater binding capacity, facilitate the antibodies to immobilize on them.
Incubate with a protein-containing solution, binding to the remaining binding surfaces of the well, reducing background interference.
Add mouse brain nuclear fraction lysate. The target protein in the lysate binds specifically to the monoclonal antibodies.
Add unlabeled polyclonal antibodies, recognizing and binding to epitopes on the protein, which is bound to primary monoclonal antibodies on the electrode. Add specific secondary antibodies labeled with ruthenium complex, binding to the unlabeled antibodies. Add a suitable buffer containing surfactant — providing a suitable ECL generation environment — and tripropylamine, TPA.
Place the multi-well plate in the ECL detection system. Upon the application of potential across the electrodes, the ruthenium complex bound to the antibodies gets oxidized. Concurrently, the TPA gets oxidized and spontaneously loses a proton.
The resulting TPA radical reduces the oxidized ruthenium complex to its excited state. Upon relaxation to its ground state, the ruthenium complex emits a photon detected by the photodetector.
The measured light intensity is representative of the lysate's specific protein concentration.
