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. 2013 Apr 2;5(1):8. doi: 10.1186/1866-1955-5-8

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Copyright © 2013 Schutzius et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Assay condition optimization for detection of human FMRP by time-resolved Förster’s resonance energy transfer. (A) Purified recombinant FMRP (5 ng) in a 1 ng/μl concentration were analyzed by two different time-resolved Förster’s resonance energy transfer (TR-FRET) immunoassays using an antibody combination detecting a N-terminal and a C-terminal FMRP epitope (N-C, Mab2160-Tb + SigmaF4055-d2) or a combination of antibodies in which both detect N-terminal epitopes (N-N, Mab2160-Tb + M03-d2). N-N antibody combination resulted in stronger signal over background detection. (B) Antibody titer optimization for N-C and N-N combination for detection of purified recombinant FMRP (5 ng). (C) Optimization of TR-FRET assay conditions for time and temperature of incubation. Both assays performed best at room temperature incubation overnight (≥20 hours). All values presented as percentage signal over assay buffer background. All data and error bars represent averages and standard deviations of triplicates.