hahn lab

affinity reagent biosensors: FRET dual chain

 

In this Rac1 biosensor an “affinity reagent” derived from Pak1 (PBD) binds specifically to the active form of the GTPase. The biosensor uses FRET as a ‘readout’ of GTPase binding.

Initial description of the approach

Kraynov, V.S., Chamberlain, C.E., Bokoch, G.M., Schwartz, M. A., Slabaugh, S. and Hahn, K.M. Localized Rac activation dynamics visualized in living cells.  Science, 290: 333-337, 2000. Online article | PDF


Additional applications

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, 461: 99-103, 2009. PMC2885353. Online article | Free PMC article


Detailed methods

Chamberlain, C.E., Kraynov, V. and Hahn, K.M. Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol., 325: 389-400, 2000. Online article | PDF

Hodgson, L. Nalbant, P. Shen, F., and Hahn, K. Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol., 406:140-156, 2006. Online article | PDF
*Note: this article is focused on dye-based biosensors, but includes a method to correct for bleaching of any fluorophore.

Hodgson, L., Pertz, O., and Hahn, K.M. Design and Optimization of genetically encoded fluorescent biosensors: GTPase biosensors. Methods Cell Biol, 85:63-81, 2008. [Link downloads PDF] Online article
*Note: this older methods article contains a useful description of biosensor image analysis.

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