Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board

1. MeCP2-ECLIA protocol

1. Preparation of washing solution, blocking buffer, and assay diluent solution (day 1)
   1. Add 500 µL of Tween 20 to 1 L of PBS to prepare a 0.05% Tween 20 in PBS solution, mix vigorously and label as “washing solution”.
      NOTE: Ensure that only freshly prepared washing buffer is used.
   2. Prepare a blocking solution of 3% blocker A (Table of Materials) in phosphate-buffered saline (PBS). Mix by gentle stirring, filter sterilize and keep them in the fridge until use for a maximum of two weeks.
   3. Add 5 mL of blocking solution to 10 mL of PBS to prepare assay diluent solution (1% blocker A in PBS).
2. Coating of high bind plates
   1. Take out a 96-well multi-array single-spot high bind plate.
      NOTE: High bind plates have a greater binding capacity and therefore a larger dynamic range than standard plates with hydrophobic surfaces.
   2. Thaw the monoclonal mouse anti-MeCP2 antibody on ice and mix 0.67 µL of the antibody with 4 mL of PBS (1:6,000 antibody dilution in PBS). Vortex the antibody solution to mix well and label the tube as “coating solution”.
   3. Carefully dispense 25 µL of coating solution in the bottom corner of each well using a multichannel pipettor; this is called the solution coating method. Tap the 96-well plate gently on each side to ensure that the coating solution covers the bottom of each well.
   4. Seal the plate with an adhesive foil and incubate the plate in the fridge at 4 °C overnight (12−16 h).
3. Blocking (day 2)
   1. Take out the plate from the fridge and remove the foil.
   2. Remove the antibody coating solution by flicking it into the waste basket and tap the plate on a paper towel to remove all the coating solution from the wells.
   3. Add 125 µL of blocking solution per well. Seal the plate again and place it on an orbital microplate shaker.
   4. Incubate the plate for 90 min at room temperature with constant shaking at 800 rpm.
4. Preparations of standards and samples
   1. During the incubation time, prepare the MeCP2 and/or TAT-MeCP2 protein standards and various samples.
      NOTE: Lysis buffer used for standard dilution must be the same as that used in the analyzed samples.
   2. Take out one vial of MePC2 and/or TAT-MeCP2 protein stock solution (250 µg/mL), mouse brain lysates, and HDF lysates from -80 °C. Thaw them on ice.
   3. Dilute the standard stock solution (MeCP2 and/or TAT-MeCP2) in clean tubes according to Table 1.
   4. Dilute the samples in lysis buffer as follows: 1−20 µg of mouse brain lysate per 25 µL of lysis buffer, and 0.25−1 µg of HDF lysate per 25 µL of lysis buffer. Prepare enough volume of each sample to carry out analysis in triplicate.
5. Adding the samples and standard solutions
   1. Remove the blocking solution by flicking it into the waste basket and tap the plate on a paper towel to remove all the blocking solution from the wells.
   2. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   3. Add 25 ul of standards and samples to the bottom corner of the well using a single-channel pipette.
   4. Seal the plate and incubate the plate for 4 h at room temperature with constant shaking at 800 rpm.
6. Unlabeled detection antibody
   1. Thaw the polyclonal rabbit anti-MeCP2 antibody on ice. Dilute the antibody 1:6,000 in assay diluent solution.
   2. Remove the standards and samples by flicking it into the waste basket and tap the plate on a paper towel.
   3. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   4. Add 25 µL of unlabeled detection antibody to each well with the multichannel pipettor. Seal the plate and incubate it for 1 h with constant shaking at 800 rpm at room temperature.
7. Specific conjugated antibody
   1. Take out the specific secondary antibody (Table of Materials) from the fridge and place it on ice. Dilute the antibody 1:666.67 in assay diluent solution and mix gently.
   2. Remove the free unlabeled secondary antibody by flicking it into the waste basket and tap the plate on a paper towel.
   3. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   4. Add 25 µL of specific conjugated antibody (Table of Materials) to each well with the multichannel pipettor. Seal the plate and incubate for 1 h with constant shaking at 800 rpm at room temperature.
8. Reading the plate
   1. Remove the free conjugated antibody (Table of Materials) by flicking it into the waste basket and tap the plate on a paper towel.
   2. Wash the plate 3x with 150 µL of washing solution.
   3. Add 150 μL of 1x Tris-based Gold read buffer (Table of Materials) with surfactant containing tripropylamine as a co-reactant for light generation to the plate. Avoid any air bubbles by using reverse pipetting techniques.
   4. Place the plate on the microplate detection platform (Table of Materials) and start the measurement immediately. Use the settings for 96-well plate acquisition.
   5. Capture the electrochemiluminescence signals by a built-in CCD camera in an electrochemiluminescence detection system (Table of Materials) and record the signal counts, which correspond to relative light units (RLU) and are directly proportional to the intensity of light.
      NOTE: Upon electrochemical stimulation, the ruthenium label bound to the carbon electrode emits luminescence light at 620 nm. Analyze data with the instrument-accompanied software (Table of Materials).

Table 1: Standard series from 0 to 1,800 ng/mL.