Figure 4.

Strength of the single molecule binding force between six consecutive histidine molecules (His₆) tag and Ni²⁺. (a) His₆ coated tip was used to probe Ni²⁺/NTA agarose beads immobilized on a His₆ functionalized glass coverslip. Tip and bead were brought in contact (approach; arrow pointing down) by means of the piezoelectric element and then taken apart (retract, arrow pointing up). The drawing in (a) is not to scale. (b) The retraction part of a typical force-distance (extension) curve using an experimental design in (a). There are several events indicating unbinding/rupture of His₆ -Ni²⁺/NTA. The very last events (arrow) were used in analysis of a single unbinding event, since it is possible that the other steps are a nonlinear convolution of multiple unbinding events. The dashed line indicates zero force. (c) Distributions of the rupture forces for His₆ -Ni²⁺/NTA single bonds. Retraction velocity, 1.6 μm/s. Arrowhead indicates the mean value.