As shown in Figure 1, the elution in R2 amplifies earlier than in Round 1. This could be an increase in binding sequences, but the more likely explanation is an inadequate wash volume. The gel image of W3 below (Figure 2) shows that there are many nucleic acid sequences that are not being washed away effectively.
Figure 2 shows that a large portion of the pool is still not being separated from the protein through consecutive washes. To resolve this problem, the wash volume will be increased in the next round. In addition, negative selection will be performed in R3, in order to reduce background binders. Finally, the incubation time between washes will be lengthened in order to allow all of the nonbinders to wash away from the target.
The concentrations at the end of R1 and R2 were 58.6 uM and 100.9 uM, respectively. This increase in concentration is a good sign, as it may indicate an increase in the concentration of binding species.