Streptavidin affinity chromatography

Gretch, D.R., Suter, M., and Stinski, M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins. Anal. Biochem. 163, 270-277. 

FIGURE 6.35 Schematic of the experimental protocol for streptavidin affinity chromatography. (1) The channel is initially filled at room temperature with a suspension of biotinylated, PNIPAAm-coated beads (100 nm). (2) The temperature in the channel is then raised to 37°C, and the beads aggregate and adhere to the channel walls. (3) Buffer is then pumped through the channel (the presence of flow is indicated in this schematic by an arrow), washing out any unbound beads. (4) A fluorescently labeled streptavidin sample (2.5 pM) is then introduced into the flow stream. (5) Streptavidin binds to the beads, and any unbound streptavidin is washed out of the channel. (6) Finally, the temperature is reduced to room temperature, leading to the breakup of the bead aggregates. Beads, bound to labeled streptavidin, elute from the channel [203], Reprinted with permission from the American Chemical Society. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

For affinity chromatography, avidin or streptavidin are coupled to supports using Protocol 3.6.2. Biotin is covalently bound to proteins or other primary amino groups bearing molecules using its N-hydroxsuccinimide ester. ... 

A wide range of immobilization chemistries are commercially available in conjunction with Sepharose beads. We have investigated a limited subset of these possibilities which include direct, nonoriented immobilization via Schiff s base chemistry, oriented nonco-valent immobilization via immobilized metal affinity chromatography resins and oriented noncovalent immobilization via biotin-streptavidin binding. At present we favor direct, covalent attachment of proteins via primary amines since it is highly efficient (typically better than 85% yield), minimizes leaching and provides the best NMR results (Figure 6.2). 

Affinity chromatography of streptavidin was performed on a PET chip. The microchannel was first filled with the dual-modified latex beads (as shown in Figure 6.3). The biotinylated beads were surface-modified with a temperature-sensitive polymer, poly(N-isopropylacrylamide (PNIPAAm, 11 kDa). When the temperature was raised above the lower critical solution temperature (LCST) of PNIPAAm, the beads aggregated and adhered to the channel wall, because of a hydrophilic-to-hydrophobic phase transition. Then streptavidin from a sample solution was captured by these adhered biotinylated beads. Thereafter, when the temperature was reduced below the LCST, the beads dissociated and eluted from the channel wall together with the captured streptavidin [203],... 

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