Methods for Detecting Protein–DNA Interactions

The chromatin immunoprecipitation (ChIP) method can be used to monitor transcriptional regulation through histone modification (epigenetics) or transcription factor–DNA binding interactions. The ChIP assay method allows analysis of DNA–protein interactions in living cells by treating the cells with formaldehyde or other crosslinking reagents in order to stabilize the interactions for downstream purification and detection. Performing ChIP assays requires knowledge of the target protein and DNA sequence that will be analyzed, as researchers must provide an antibody against the protein of interest and PCR primers for the DNA sequence of interest. The antibody is used to selectively precipitate the protein–DNA complex from the other genomic DNA fragments and protein–DNA complexes. The PCR primers allow specific amplification and detection of the target DNA sequence. Quantitative PCR (qPCR) technique allows the amount of target DNA sequence to be quantified. The ChIP assay is amenable to array-based formats (ChIP-on-chip) or direct sequencing of the DNA captured by the immunoprecipitated protein (ChIP-seq).

The DNA electrophoretic mobility shift assay (EMSA) is used to study proteins binding to known DNA oligonucleotide probes and can be used to assess the degree of affinity or specificity of the interaction. The technique is based on the observation that protein–DNA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis. Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also referred to as a gel shift or gel retardation assay. Adding a protein-specific antibody to the binding components creates an even larger complex (antibody–protein–DNA), which migrates even slower during electrophoresis. This is known as a “supershift”, and it can be used to confirm protein identities. Until conception of the EMSA, protein–DNA interactions were studied primarily by nitrocellulose filter–binding assays using radioactively labeled probes.

Traditionally, DNA probes have been radiolabeled with ³²P by incorporating an [γ-³²P]dNTP during a 3' fill-in reaction using Klenow fragment or by 5' end labeling using [γ-³²P]ATP and T4 polynucleotide kinase. Following electrophoresis, the gel is exposed to X-ray film to document the results. The Thermo Scientific LightShift Chemiluminescent EMSA Kit is a non-radioactive assay that provides robust and sensitive performance. The kit includes reagents for setting up and customizing DNA-binding reactions, a control set of DNA and protein extract to test the kit system, stabilized streptavidin–HRP conjugate to probe for the biotin-labeled DNA target, and an exceptionally sensitive chemiluminescent substrate module for detection.

Chemiluminescent EMSA of four different DNA–protein complexes. Biotin-labeled target duplexes ranged in size from 21–25 bp. The Oct-1, AP1 and NF-κB transcription factors were derived from HeLa nuclear extract. EBNA-1 extract is provided as a control in the LightShift Chemiluminescent EMSA Kit. Unlabeled specific competitor sequences (where used) were present at a 200-fold molar excess over labeled target. X-ray film exposure times for each system ranged from 2 minutes for EBNA, Oct-1 and AP1, and 5 minutes for NF-κB.

Pull-down assays are used to selectively extract a protein–DNA complex from a sample. Typically, the pull-down assay uses a DNA probe labeled with a high affinity tag, such as biotin, which allows the probe to be recovered or immobilized. A biotinylated DNA probe can be complexed with a protein from a cell lysate in a reaction similar to that used in the EMSA and then used to purify the complex using agarose or magnetic beads. The proteins are then eluted from the DNA and detected by western blot or identified by mass spectrometry. Alternatively, the protein may be labeled with an affinity tag, or the DNA–protein complex may be isolated using an antibody against the protein of interest (similar to a supershift assay). In this case, the unknown DNA sequence bound by the protein is detected by Southern blotting or through PCR analysis.

A hybrid of the DNA pull-down assay and enzyme-linked immunosorbent assay (ELISA), microplate capture assays use immobilized DNA probes to capture specific protein–DNA interactions and confirm protein identities and relative amounts with target specific antibodies. Typically, a biotinylated DNA probe is immobilized on the surface of a 96- or 384-well microplate coated with streptavidin. A cellular extract is prepared in binding buffer and added for a sufficient amount of time to allow the putative binding protein to bind to the oligonucleotide. The extract is then removed and each well is washed several times to remove nonspecifically bound proteins. Finally, the protein is detected using a specific antibody labeled for detection. This method can be extremely sensitive when performed with enzyme-labeled antibodies and a chemiluminescent substrate, detecting less than 0.2 pg of the target protein per well. The microplate format is efficient and compatible with high-throughput analysis, allowing statistical mutational and activation assays to be performed. This method may also be utilized for oligonucleotides labeled with other tags, such as primary amines that can be immobilized on microplates coated with an amine-reactive surface chemistry.

Reporter assays provide a real-time in vivo readout of translational activity for a promoter of interest. Reporter genes are fusions of a target promoter DNA sequence and a reporter gene DNA sequence. The promoter DNA sequence is customized by the researcher and the reporter gene DNA sequence codes for a protein with detectable properties such as firefly luciferase, Renilla luciferase or alkaline phosphatase. These genes produce enzymes only when the promoter of interest is activated. The enzyme, in turn, catalyzes a substrate to produce either light, a color change, or other reaction that can be detected by spectroscopic instrumentation. The signal from the reporter gene is used as an indirect determinant for the translation of endogenous proteins driven from the same promoter.

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