Fn isolated from human plasma (see Text S1 ) was doubly labeled with Alexa 488 and Alexa 546 (Molecular Probes, http://probes.invitrogen.com/ ) as FRET donors and acceptors, respectively, using two different labeling schemes based on established protocols [24 (link),25 (link)]. To produce cys/cys Fn-DA, Fn was site-specifically labeled exclusively on buried cysteines within modules III7 and III15 of each dimer arm. Isolated plasma Fn at ∼1 g/l in PBS was denatured in an equal volume of 8 M GdnHCl and incubated for 2 h with a 15-fold molar excess each of Alexa 546 maleimide and Alexa 488 maleimide. Fn-DA was separated from free dye by size exclusion chromatography (PD-10 Sephadex, Amersham, http://www.amersham.com/ ). Western blot analysis was used to determine the point at which Fn fragments contaminated the eluent (data not shown). Fn was also labeled to produce amine/cys Fn-DA with donors and acceptors located on random amines and cysteines, respectively. Fn at ∼1 g/l in PBS was denatured in an equal volume of 8 M GdnHCl and incubated for 1 h with a 30-fold excess of Alexa 546 maleimide. Acceptor-labeled Fn was separated from free dye by overnight dialysis (Slide-a-lyzer dialysis cassette, 10,000 MW cutoff; Pierce, http://www.piercenet.com/ ) with three changes of amine labeling buffer (PBS with 0.1 M NaHCO3 [pH 8.5]). Subsequently acceptor-labeled Fn was incubated with a 70-fold excess of Alexa 488 succinimidyl ester for 1 h. Fn-DA was separated from free dye using a PD-10 column equilibrated with PBS. The labeling ratio of donors to acceptors per Fn dimer was determined by measuring the absorbances of Fn-DA at 280, 496, and 556 nm and using published extinction coefficients for dyes and Fn. Fn-DA was stored with 10% glycerol at −20 °C until needed and used within 5 d of thawing and storage at 4 °C.
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