Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate
Published: April 30, 2023
Abstract
Source: Steinkellner, H., et al., An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants. J. Vis. Exp. (2020).
In this video, we demonstrate the electrochemiluminescence immunoassay for the quantitation of target protein levels in different samples. This assay is based on the luminescence produced in the electrochemical reaction. The intensity of light emitted is proportional to the quantity of the target protein in the sample.
Protocol
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 Preparation of washing solution, blocking buffer, and assay diluent solution (day 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 p…
Divulgations
The authors have nothing to disclose.
Materials
| TAT-MeCP2 fusion protein | In-house production | Cell treatment | |
| MSD Blocker A | Meso Scale Diagnostics | R93BA-4 | Blocker |
| Tween 20 | Sigma-Aldrich | P9416 | Washing solution |
| Polyclonal Anti-MeCP2, produced in rabbit | Eurogentec S.A. | custom-designed | Antibody |
| Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin | Sigma-Aldrich | M6818-100UL; RRID:AB_262075 | Antibody, Coating solution |
| Multi-Array 96-well Plate | Meso Scale Diagnostics | L15XB-3/L11BX-3 | MeCP2 ECLIA protocol |
| Secondary AB, Rabbit, anti-MeCP2 | Eurogentec S.A. | custom | Antibody |
| Secondary AB, Rabbit, anti-MeCP2 | Merck | 07-013 | Antibody |
| MeCP2 (Human) Recombinant Protein (P01) | Abnova Corporation | H00004204-P01 | Cell treatment |
| Gold Read Buffer T (1x) with surfactant | Meso Scale Diagnostics | R92TG | MeCP2 ECLIA protocol |
| MSD SECTOR Imager 2400 | Meso Scale Diagnostics | I30AA-0 | MeCP2 ECLIA protocol |
| Discovery workbench 4.0 | Meso Scale Discovery | Software | |
| Dulbecco’s PBS (sterile) | Sigma-Aldrich | D8537-500ML | Sample preparation, Washing solution, Coating solution |
| EDTA | Sigma-Aldrich | EDS | Extraction Buffer |
Tags
Citer Cet Article
Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate. J. Vis. Exp. (Pending Publication), e21333, doi: (2023).