Label-free binding approaches

Co-immunoprecipitation is a common technique to enrich protein interaction complexes. This method uses an antibody to bind a known protein, which in turn has known or unknown binding partners. Shotgun proteomics is then performed to determine the enriched proteins. Quantitative MS is used to validate bound proteins and measure binding stoichiometry for proteins in the complexes prepared via IP (127,128). Quantitation approaches used include SILAM (129), label-free (130), and AQUA peptides with a normalization step (131). 

Diakowski and Kraatz also used [Fe(CN)g]" to study mismatches in a 25 base pair ODN hybridization experiment [144], The negative feedback was greatest for fully matched strands and least for mismatches near the ends of the strands. Their data were reported in terms of the effective rate constant describing the faradaic reaction at the substrate as extracted from feedback approach curves (Chapter 5). Interestingly, a threefold difference between the rate constants at various surfaces with matched/mismatched pairs was observed that was greatly increased to a factor of about 20-fold in the presence of Zn +. This effect is due to the modulation of the charge density of the DNA by the binding of Zn + [138] and appears to be a useful means to enhance the performance of such label-free DNA sensors. 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

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