Success with equations
I have solved the issue of excess versus limiting DNA in bandshift reactions. It turns out that my confusion arose because of the two distinctly different ways that the equation for deriving equilibrium binding constant can be derived: the common (overly simplified) equation for calculating Kd (the dissociation constant, which reflects a protein’s affinity for a DNA site) ignores the important qualifiers of the protein and DNA concentrations in the reaction. Unfortunately, the Kd equation cannot be easily illustrated in this blogger post due to my inability to write equations in a blog, so I will add the equation (and its derivatives) tomorrow when I have time to draw them out and edit this post.
The bottom line is that all of the bandshift experiments I have conducted have been informative, and now I can confidently proceed to the next step of measuring accurate Kd values for CRP binding to different CRP sites. Also, tomorrow we will test a second prep of His-tagged Sxy to see if once again CRP has been co-purified along with Sxy. If positive (ie. CRP was pulled down with His-Sxy), we can easily test how much salt needs to be added to His-Sxy to prevent the co-purification of CRP; the amount of salt needed to block the interaction will give us a sense of the strength of affinity between the two proteins.