Note re Rac biosensors from Hahn Lab

Our original Rac biosensor was published in 2000 ((Kraynov et al. Science, 290:333-337, 2000)). At that time, optimization of mutants for GFP FRET had just begun, and we found that use of FRET between GFP and an organic dye was better than that between two mutants of GFP. That has since changed. Our lab has produced Rac sensors that are fully genetically encoded, and far more sensitive than our first-published Rac sensor. These new sensors have not yet been published, but will be submitted and made available within roughly 6 months.

The published sensor is more difficult to make and use than many current biosensors; one of the components must be made by covalent dye attachment, and must then be loaded into cells via microinjection or some other means other than transfection. Most importantly, the extent of FRET is relatively weak, so that careful image corrections and controls must be used.

We will of course continue to supply the published vectors from the original Kraynov paper. In some cases, it is possible to see GTPase activation simply by following the changing localization of the biosensor, without using FRET (i.e. Srinivasan et al. JCB, 160(3):375-85, 2003). For this purpose one could use some of the constructs we have deposted at Addgene.

Some hints and protocols for  use of the original Rac sensor with FRET can be found in a Methods article published shortly after the original article:

Chamberlain, C. E., V. Kraynov and K. M. Hahn. Imaging Spatiotemporal Dynamics of Rac Activation in vivo with FLAIR. Methods in Enzymology, 325:389-400, 2000.

Link to PDF of this article

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