Measuring Low-Picomolar Apparent Binding Affinities by Minigel Electrophoretic Mobility Shift
Date
2019
Authors
Lewis, Karen A.
Altschuler, Sarah E.
Wuttke, Deborah
Journal Title
Journal ISSN
Volume Title
Publisher
Humana Press
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 K D,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.
Description
Keywords
EMSA, DNA binding protein, binding assay, high-affinity binding, native gel, Chemistry and Biochemistry
Citation
Lewis, K. A., Altschuler, S. E., & Wuttke, D. S. (2019). Measuring low-picomolar apparent binding affinities by minigel electrophoretic mobility shift. Methods in Molecular Biology, 1855.