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Methods for Detecting Protein-DNA Interactions

Proteins interact with DNA through electrostatic interactions (salt bridges), dipolar interactions (hydrogen bonding, H-bonds), entropic effects (hydrophobic interactions) and dispersion forces (base stacking). These forces contribute in varying degrees to proteins binding in a sequence-specific or non-sequence specific manner. Understanding how proteins interact with DNA, determining what proteins are present in these protein-DNA complexes and identifying the nucleic acid sequence (and possible structure) required to assemble these complexes are vital to understanding the role these complexes play in regulating cellular processes. A number of laboratory techniques have been developed to study the complex interactions of proteins with DNA, each with a unique history, varying utility and distinct strengths and weaknesses.

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Protein Interaction
Technical Handbook

Chromatin Immunoprecipitation (ChIP) Assays

The 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 which will be analyzed, as researchers must provide an antibody against the protein of interest and PCR primers for the DNA sequence if 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).

Strengths:
  • capture a snapshot of specific protein-DNA interactions as they occur in living cells
  • quantitative when coupled with qPCR analysis
  • ability to profile a promoter for different proteins
Limitations:
  • researcher needs to source ChIP grade antibodies
  • requires designing specific primers
  • difficult to adapt for high-throughput screening.

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ChIP Assay Procedure and Essential Tools

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Chromatin IP Assay Kits

DNA Electrophoretic Mobility Shift Assay (EMSA)

The 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 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.

Strengths:
  • detect low abundance DNA binding proteins from lysates
  • test binding site mutations using many probe configurations with the same lysate
  • test binding affinity through DNA probe mutational analysis
  • non-radioactive EMSA possible using biotinylated or fluorescently labeled DNA probes
Limitations:
  • analyze protein-DNA interactions in vitro
  • difficult to quantitate
  • need to perform supershift assay with antibody to be certain of protein identity in a complex

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Methods for Labeling Nucleic Acids

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LightShift Chemiluminescent EMSA Kit

Biotin 3' End DNA Labeling Kit

DNA Pull-down Assay

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.

Strengths:
  • enrichment of low abundant targets
  • end-labeled DNA can be generated by several methods
  • isolation of intact complex
  • compatible with immunoblotting and mass spectrometry analysis
Limitations:
  • long DNA probes can show significant non-specific binding
  • requires very specific antibodies for native proteins
  • nuclease-free conditions required
  • assay must be performed in vitro

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Methods for Labeling Nucleic Acids

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Immunoprecipitation and Pull-Down Kits

Biotin 3' End DNA Labeling Kit

 

Microplate Capture and Detection Assay

A hybrid of the DNA-pulldown assay and 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 96- or 384-well microplates 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 0.2pg of the target protein per well. The microplate format is efficient and high throughput compatible, 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.

Strengths:
  • the use of ELISA-based technology increases speed and throughput
  • compatible with drug screening
  • possible to optimize sensitive non-radioactive assays
Limitations:
  • requires antibodies with affinity for DNA-bound native proteins (i.e., supershift antibodies)
  • data only provides relative changes in transcription factor-DNA affinity or abundance
  • assay kits are available for only a few targets

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Chemiluminescent Transcription Factor Assay Kits

Reporter Assays

Reporter assays provide a real-time in vivo read-out 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.

Strengths:
  • in vivo monitoring
  • capture real-time data
  • powerful tool for mutational analysis of promoters
  • amendable to high-throughput screening
Limitations:
  • uses exogenous DNA
  • does not address changes due to genomic sequences near the promoter of interest
  • artifacts due to gene dosage can occur.

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Methods for Detecting Protein-RNA Interactions

Luciferase Reporters

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Luciferase Reporter Assays

References

Selected References for Studying Protein-DNA Interactions:
  1. Evertts A.G., et al. (2010). Modern approaches for investigating epigenetic signaling pathways. J Appl Physiol. Jan 28. [Epub ahead of print]
  2. Georges, A.B., et al. (2010). Generic binding sites, deneric DNA-binding domains: Where does specific promoter recognition come from? FASEB Journal, 24: 346-356.
  3. Griffiths, Anthony J. F., et al., eds (2000). "Genetics and the Organism: Introduction". An Introduction to Genetic Analysis (7th ed.). New York: W. H. Freeman.
  4. Halford, S.E. and Marko, J. (2004). How do site specific DNA-binding proteins find their target? Nuc. Acid Research. 32(10): 3040-3052.
  5. Hartl, Daniel L., et al. (1988). Basic Genetics, Boston: Jones and Bartlett Publishers, Inc.
  6. Kress, C., et al. (2010). Epigenetic modifications in 3D: Nuclear organization of the differentiating mammary epithelial cell. J Mammary Gland Biol Neoplasia. Feb 10. [Epub ahead of print]
  7. Lunde, B.M. et al. (2007). RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell. Biol. 8: 479-490.

 

Written and/or reviewed by Douglas Hayworth, Ph.D.

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