Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate

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.

Retrieve the 96-well plate from the refrigerator, and remove the foil. Remove the antibody-coating solution from the wells by flicking it into a waste container. Then, tap the plate on a paper towel to remove any remaining solution.

Next, add 125 microliters of blocking solution to each well. Then, seal the plate again with adhesive foil. Place the plate on an orbital microplate shaker, set at 800 RPM, and incubate it for 90 minutes at room temperature.

Thaw on ice, one vial of MeCP2 or TAT-MeCP2 protein stock solution, mouse brain lysates, and HDF lysates. Dilute the protein stock solution in clean tubes, as described in Table 2 of the manuscript.

Next, dilute the lysate samples. For the mouse brain lysates, use 1 to 20 micrograms per 25 microliters of lysis buffer. For the HDF lysate, add 0.25 to 1 microgram per 25 microliters of lysis buffer.

After the 96-well plate has been incubated for 90 minutes, remove the blocking solution from the wells by flicking it into a waste container. Then, tap the plate on a paper towel to remove any remaining solution. Then, wash the plate 3 times with 150 microliters of washing solution, by adding the washing solution and immediately removing it.

Add 25 microliters of standards and samples to the wells of the 96-well plate. Seal the plate and incubate it for another four hours at room temperature, with constant 800 RPM shaking.

Thaw the unlabeled detection antibody, polyclonal rabbit anti-MeCP2, on ice. Using assay diluent solution, dilute the antibody at a ratio of 1:6000. When the 96-well plate has finished incubating on the shaker, remove the standards and samples by flicking the plate into a waste container. Then, tap the plate on a paper towel to remove any remaining solution.

Wash the plate 3 times with 150 microliters of washing solution by adding the washing solution, and immediately removing it. Add 25 microliters of unlabeled detection antibody solution to each well with the multichannel pipetter. Seal the plate and incubate it for 1 hour at room temperature with constant shaking at 800 RPM.

Obtain the specific secondary antibody from the refrigerator, and place it on ice. Dilute the secondary antibody in assay-diluent solution and mix gently.

To remove the free un-labeled detection antibody from the 96-well plate, flick it into the waste container, and then, tap the plate on a paper towel. After washing the plate 3 times, as previously described, add 25 microliters of secondary antibody to each well with the multichannel pipettor. Seal the plate and incubate it for 1 hour at room temperature with constant shaking at 800 RPM.

Remove the free secondary antibody by flicking it into the waste container, and tapping the plate on a paper towel, and then, wash the plate three times as previously described.

To each well of the plate, add 150 microliters of 1X tris-based Gold read buffer with surfactant, containing tripropylamine as a co-reactant for light generation.

To avoid producing air bubbles, use reverse pipetting techniques. Place the 96-well plate on the microplate platform with an electrochemiluminescence detection system.

Using the built-in CCD camera, immediately begin capturing data. Record the signal counts which correspond to relative light units and are directly proportional to the intensity of light.