Note re new Rac and Cdc42 Biosensors

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 more sensitive than our first-published Rac sensor. These new sensors are briefly described in Machacek, M., Hodson, L., Welch, C., Elliot, H., Pertz, O., Nalbant, P., Abell, A., Johnson, G., Hahn, K.M. and Danuser, G. Coordination of Rho GTPase activities during cell protrusion. Nature 2009, in press. Detailed characterizations will be published. A new cdc42 biosensor of similar design will also be published soon.

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 fluorescent 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 deposited 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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