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An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Published on: November 29, 2016
On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing
Alexander V Stoyanov1, Z Hugh Fan, Champak Das
1VPB, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA. stoyanov@purdue.edu
Abstract:
Noncovalent fluorescent dyes are widely used for protein quantification and postcolumn detection in electrophoretic separations and recently some attempts to separate the precolumn labeled proteins using isoelectric focusing (IEF) have been made. In the present study, the possibility of applying the technique of protein labeling with noncovalent dyes for IEF is investigated. We found that fluorescent signal emitted by NanoOrange dye increases essentially in presence of carrier ampholyte (CA) components, which makes problematic a reliable protein detection in CA environment. Since in an isoelectric focusing mode the CA species are present in much greater concentration than the concentrations of fractionated proteins, the method of protein labeling with NanoOrange is not suitable for precolumn labeling and cannot be used for CA-IEF, at least without more detailed study of the dye-protein interaction mechanism.
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