蛋白质表达及标记

aqi96

This protocol refers to the biosensor described in the following paper: Nalbant, P., Hodgson, L., Kraynov, V., Toutchkine, A., and Hahn, K.M. Activation of Endogenous Cdc42 Visualized in Living Cells. Science, 305:1615-1619, 2004.

The following procedures are very detailed, but are actually quite straightforward. This biosensor is made by expressing a protein and then covalently attaching a dye. We have published synthesis of one such dye (Toutchkine, A., Kraynov, V., and Hahn, K.M. Solvent-Sensitive Dyes to Report Protein Conformational Changes in Living Cells, J. Amer. Chem. Soc., 125:4132-4145, 2003.).  Please contact Klaus Hahn if you intend to make the biosensor, as we are improving the dyes and will likely have more recent improved versions.



Contents:
1. protein expression and purification
2. labeling with I- SO IAA dye
3. generic dye labeling protocol
4. note re photobleach correction