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Single- and dual-parameter FRET kinase probes based on pleckstrin

Abstract

Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h.

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Figure 1: DNA fragments containing the coding sequences for EYFP, GFP2 and a dummy fragment are ligated together to create pYG.
Figure 2: The pleckstrin coding region of KCP-1 is amplified with primers containing the indicated restriction sites.
Figure 3: Oligonucleotides, in this case encoding the PKA consensus sequence kemptide, are annealed and phosphorylated before ligation with BbsI-opened mKCP-1.
Figure 4: An example trace from the PKA/PKC sensor KCAP-1 in N1E-115 cells.
Figure 5: Influence of expression levels on probe performance.
Figure 6

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Correspondence to Carsten Schultz.

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Brumbaugh, J., Schleifenbaum, A., Stier, G. et al. Single- and dual-parameter FRET kinase probes based on pleckstrin. Nat Protoc 1, 1044–1055 (2006). https://doi.org/10.1038/nprot.2006.177

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