Protein:DNA Interactions

Promega products for studying protein:DNA interactions include gel shift assays and the HaloCHIP™ System—a fast alternative to traditional chromatin immunoprecipitation methods. In the HaloCHIP™ method, DNA-binding proteins of interest are expressed in cells as HaloTag® fusion proteins, crosslinked to DNA and then captured using the HaloLink™ Resin, which forms a highly specific, covalent interaction with HaloTag® proteins. The resin can be stringently washed to remove nonspecifically bound DNA and protein more efficiently than co-immunoprecipitation. The crosslinks are reversed to release purified DNA fragments from the resin. No antibodies are required.

The Gel Shift Assay Systems contain target oligonucleotides, a control extract containing DNA-binding proteins, binding buffer and reagents for phosphorylating oligonucleotides. The complete Gel Shift Assay System contains five additional double-stranded oligonucleotides that represent consensus binding sites for AP1, NF-κB, OCT1, CREB and TFIID. These oligonucleotides can be end-labeled and used as protein-specific probes or as specific or nonspecific competitor DNA in competition assays.

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Protein:DNA Interactions Basics

Analysis of protein:DNA interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags. Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface. Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions.

Chromatin immunoprecipitation (ChIP) is a method used to determine whether DNA-binding proteins, such as transcription factors, associate with a specific genomic region in living cells or tissues. Cells are treated with formaldehyde to form covalent crosslinks between interacting proteins and DNA. Following crosslinking, cells are lysed, and the crude cell extracts are sonicated to shear the DNA. The DNA:protein complex is immunoprecipitated using an antibody that recognizes the protein of interest. The isolated complexes are washed and then eluted.

The standard ChIP assay requires 3–4 days for completion and requires antibodies highly specific to the protein of interest to immunoprecipitate the DNA:protein complex. HaloCHIP™ assays take 24–48 hours and do not require antibodies, generating results in less time and with fewer steps than traditional ChIP assays.

The electrophoretic mobility shift assay (EMSA) or gel shift assay is a simple method to detect DNA-binding proteins that is used widely in the study of sequence-specific DNA-binding proteins such as transcription factors. The gel shift assay is based on the observation that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides due to the increase in mass.

Gel shift assays are performed by incubating a purified protein or a complex mixture of proteins with a labeled DNA fragment containing the putative protein-binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences.