The authors have declared no conflict of interest.
Fluorogen-activating-proteins as universal affinity biosensors for immunodetection
Article first published online: 14 NOV 2013
© 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 111, Issue 3, pages 475–484, March 2014
How to Cite
Gallo, E., Vasilev, K. V. and Jarvik, J. (2014), Fluorogen-activating-proteins as universal affinity biosensors for immunodetection. Biotechnol. Bioeng., 111: 475–484. doi: 10.1002/bit.25127
- Issue published online: 21 JAN 2014
- Article first published online: 14 NOV 2013
- Accepted manuscript online: 9 OCT 2013 07:38AM EST
- Manuscript Accepted: 2 OCT 2013
- Manuscript Revised: 13 SEP 2013
- Manuscript Received: 11 JUL 2013
- NIH National Center for Research Resources. Grant Number: U54RR022241
- affinity reagents;
Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins—Protein A or Protein G—and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. Biotechnol. Bioeng. 2014;111: 475–484. © 2013 Wiley Periodicals, Inc.