Figure 3.

Surface immobilization strategies for smFRET experiments. (a) Biotin-BSA proteins bound non-specifically to the surface tether biotinylated molecules with the aid of multivalent avidin proteins (e.g. neutravidin). (b) Mixture of biotin-PEG and PEG is covalently attached to amino silanized slide surface. Biotins on the PEG can bind DNA (or protein) engineered with a biotin moiety with the help of a sandwiched neutravidin protein. PEG coating prevents the non-specific binding. (c) PEG coated surfaces can be engineered to carry Ni²⁺ or Cu²⁺ chelated on iminodiacetic acid (IDA) (or nitrilotriacetic acid (NTA)) groups that bind efficiently to 6× His-tagged proteins while retaining functional activity. (d) Using dimyristoyl phospatidylcholine (DMPC) at room temperature, vesicles selectively permeable to small molecules can be used to trap biomolecules. A fraction of biotinylated lipid allows specific tethering of the vesicles to the biotin-PEG surface. D and A labels stand for donor and acceptor dyes.