Abstract
The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.
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Acknowledgements
This material is based upon works supported by the Science Foundation Ireland under Grant No. 04/RP1/B499. The sample of human ventricular heart tissue was kindly provided, with ethical approval, by Prof. Marlene Rose (Imperial College, London, UK).
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Scaife, C., McManus, C.A., Donoghue, P.M., Dunn, M.J. (2015). Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow. In: Kurien, B., Scofield, R. (eds) Detection of Blotted Proteins. Methods in Molecular Biology, vol 1314. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2718-0_15
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DOI: https://doi.org/10.1007/978-1-4939-2718-0_15
Publisher Name: Humana Press, New York, NY
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