Measuring low-picomolar apparent binding affinities by minigel electrophoretic mobility shift

Abstract

Measuring protein/DNA interactions that have apparent binding affinity constants in the low-picomolar range presents a unique experimental challenge. To probe the sequence specificity of telomere binding proteins, our laboratory has developed an electrophoretic mobility shift assay protocol that allows for the routine measurement of KD,app values in the 1–20 pM range. Here, we describe the protocol and highlight the particular considerations that should be made to successfully and reproducibly measure high-affinity interactions between proteins and single-stranded DNA.