We describe a protocol for the preparation of a family of calcium indicators for MRI. The indicators consist of superparamagnetic iron oxide nanoparticles functionalized with the calcium-sensing protein, calmodulin, and its target peptides. Calcium-dependent protein–protein interactions drive particle clustering and produce large changes in T2 relaxivity. To prepare these indicators, interacting polypeptides are purified, biotinylated and then conjugated to streptavidin-coated nanoparticles. Conjugates are isolated, mixed to obtain functional sensors and incubated in the presence or absence of calcium to test response properties. Calcium-dependent responses are assayed by dynamic light scattering and MRI. The whole series of procedures can be carried out in ∼12 h. The iron oxide-based calcium sensors discussed here are suitable for the detection of calcium concentration changes of ∼1 μM in vitro.
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This work was generously funded by the Raymond and Beverley Sackler Foundation, the McKnight Endowment Fund for Neuroscience and the National Institutes of Health (R21 EB005723 to A.J.). A.J. is a Raymond and Beverley Sackler Foundation Scholar. The authors are grateful to Atsushi Miyawaki for the Xenopus CaM D64Y E104Q template sequence.
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Atanasijevic, T., Jasanoff, A. Preparation of iron oxide-based calcium sensors for MRI. Nat Protoc 2, 2582–2589 (2007). https://doi.org/10.1038/nprot.2007.377
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