Force Spectroscopy: Cantilever Functionalization
The ligand must be immobilized on the AFM tip in order to acquire direct force measurements of ligand-receptor pairs. The techniques commonly used involve either chemi-adsorption (Moy et al., 1994) or covalent coupling of the ligand to the tip via an extended linker (Hinterdorfer et al., 1996). The linker between the tip and the ligand lends greater mobility and access to receptors on the surface being probed. The following outlines a method for functionalizing cantilever tips with a biotinylated ligand (e.g., biotin-concanavalin A) via a streptavidin/biotin linkage (see Figure). This method is advantageous since the streptavidin/biotin system has been well-characterized, is high-affinity, and the initial layer of biotin-BSA may help to mask any electrical charges on the cantilever tip that could lead to nonspecific binding.
MATERIALS
AFM cantilevers (MLCT-AUHW; Veeco), biotin-BSA (A-6043; Sigma), streptavidin (Sigma), sodium bicarbonate buffer (0.1 M, PH 8.3), phosphate-buffered saline (pH 7.4), acetone.
METHOD
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Soak cantilever for 5 min. in acetone and then UV irradiate for 15 min.
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Incubate cantilever in a 50 µl drop of biotin-BSA (0.5 mg/ml in 0.1 M sodium bicarbonate, pH 8.3) overnight at 37°C in a humidified incubator.
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Wash cantilever three times in PBS (pH 7.4) to remove unbound protein. Fix in 1% glutaraldehyde for 30 s. Wash again with PBS. At this point cantilevers can be stored in PBS for up to a week at 4°C.
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Incubate cantilever in a 50 µl drop of streptavidin (0.5 mg/ml in PBS, pH 7.4) for 10 minutes at room temperature.
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Wash cantilever three times in PBS.
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Biotinylated ligand may be coupled to the streptavidin-functionalized tip at this step (e.g., biotinylated concanavalin A; 0.5 mg/ml in PBS, 10 min incubation at room temperature).
Note:
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It is important that biotin-BSA adsorption takes place at pH 8.3 or higher, as the basic conditions seem to facilitate BSA adsorption to the cantilever.
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Kits for biotinylating the ligand of interest are readily available from Pierce Chemical Company.
 4-AFM Measurements of the Streptavidin-Biotin Interaction
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