Affinity chromatography biotinylated

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

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. 

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],... 

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. 

Figure 18 Glycoprotein-enrichment strategies for proteomics. (a) Lectin affinity chromatography (b) chemoenzymatic modification of 0-GlcNAc-modified proteins with biotinylated probes for proteomic analysis after avidin chromatography.

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