Avidin 2-iminothiolane

Figure 23.4 Avidin may be modified with 2-iminothiolane to produce sulfhydryl groups. Subsequent reaction with a maleimide-activated enzyme produces a thioether-linked conjugate.

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

In 2001, Van Orden et al. reported the first biologically relevant FRET investigation using QDs as energy donors.83 They conjugated biotinylated bovine serum albumin (bBSA) to water-soluble CdSe-ZnS-TA QDs via a thiol linkage with 2-iminothiolane. The acceptor was streptavidin covalently labeled with tetra-methylrhodamine (TMR). Once the avidin-biotin interaction took place, a decrease in the QD luminescence and an increase in the TMR luminescence were observed, which was attributed to FRET between QD and TMR. Although no time-resolved fluorescence experiments were carried out and no estimates for the separation distance... 

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