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Procedure and Key Optimization Strategies for an Automated CapillaryElectrophoretic-based Immunoassay Method
Citation:
Nelson, G., J. Currier, AND B. Chorley. Procedure and Key Optimization Strategies for an Automated CapillaryElectrophoretic-based Immunoassay Method. JoVE, Somerville, MA, 2017.
Impact/Purpose:
This is the methods video associated with the methods article that was recently approved (ORD-018844). The linked article is a methods paper based on a general protocol for a capillary-based immunoassay that was a key tool for a recent publication from our lab (Currier et al. PLOS ONE 2016; PMID: 27195669). While the research presented in this manuscript was optimization data performed as a result of this paper, which supported ACE PEP research, the method itself is a general tool and potential replacement technology for standard Western blot immunoassays of proteins in prepared samples. Therefore the method has wide applicability to RAP-related research. Of note, the target journal for this paper is the Journal of Visualized Experiments (JoVE). If accepted, the protocol will be published as a visual protocol and highlight the EPA authors (Chorley, Guynn, and Nelson). This is therefore not only a valuable scientific reference, but highlights the technology utilized by EPA scientists to support our Agency work
Description:
New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and α-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an α-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.
URLs/Downloads:
https://www.jove.com/video/55911/procedure-key-optimization-strategies-for-an-automated-capillary