Identification of Proteins Interacting with Single Nucleotide Polymorphisms (SNPs) by DNA Pull-Down Assay

Abstract

Single nucleotide polymorphisms (SNPs) are the most abundantly found common form of DNA variation in the human genome. Many genetic association studies have proved that some of these SNPs are involved in regulating several cellular/physiological processes ranging from gene regulation to disease development. Analysis of the protein complex that binds to these SNPs is a crucial step in studying the mechanisms by which gene expressions are regulated in cis- or trans-acting manner. Commonly used techniques to determine DNA-protein interaction, such as electrophoretic mobility shift assay (EMSA), have limited value for simultaneously analyzing a large number of proteins in the complex. Furthermore, this assay is tedious and time-consuming and often requires radiolabeled probe as well as extensive optimization. Here, we describe a pull-down assay before performing the EMSA, which helps in the detection of differentially-bound protein(s) in an allele-specific manner. The assay is easy to perform and does not require radiolabeling of DNA probes. Biotinylated DNA probe bound to streptavidin beads can be complexed with protein(s) from cell nuclear lysate, nonspecific proteins were washed out, and only protein(s) having high affinity to SNP-specific DNA were detected on SDS-PAGE and identified by mass spectrometry.