Doubly labelling single protein molecules for FRET studies

4 Doubly labelling single protein molecules for FRET studies 

1 General protocol for site specific double labelling of single proteins 

Mutants containing a single cysteine is created for every putative site. The reactivity of each of these sulphydryl groups is measured using stopped-flow and monitoring (by absorbance) the production of thiolate ions released upon 

Pairs of sites are then selected which show large differences in their reaction rates (thus allowing sequential labelling) and are positioned such that the conformational change or structural element to be characterized can be monitored, with particular attention placed on the absolute distances between pairs of sites and the expected distance change upon conformational reconfiguration. 

This technique has been used to generate cold-shock protein labelled with Alexa 488 and Alexa 594 at its N- and C-termini [44]. However, it is unlikely that 

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