Filter Binding Assays

The term filter binding assay is used to describe a variety of different techniques in biochemistry, immunology, virology and molecular biology. In molecular biology a filter binding assay is used to characterize DNA–protein interactions. This particular technique is also referred to as a filter assay or a protein binding assay, and it can be used to identify and characterize both DNA-binding proteins and the DNA sequences that interact with these proteins. The filter binding assay is based on the observation that proteins, but not double-stranded DNA molecules, bind to the surface of nitrocellulose membrane filters (1, 2). Therefore, if a DNA-binding protein is incubated with a specific DNA sequence before analysis in the filter binding assay, the resulting DNA–protein complex and any excess protein will bind to the membrane filter, while uncomplexed DNA will pass through. The amount of the DNA–protein complex retained on the membrane filter can be determined by using radiolabeled DNA to form the DNA–protein complexes and to quantify the amount of radioactivity retained on the nitrocellulose membrane by scintillation counting. The filter binding assay can be very quick if a multifilter vacuum filtration unit is used.

The filter binding assay offers a simple, rapid, sensitive, and versatile assay capable of providing information about the interactions between a DNA-binding protein and a specific DNA sequence. The use of radioactive DNA makes low concentrations of DNA and protein feasible. A typical experiment involves the titration of a constant amount of radiolabeled DNA with the DNA-binding protein of interest. The amount of radioactivity retained on the membrane increases with increasing protein concentration until all the radioactive DNA that can bind to the specific DNA-binding protein is depleted from the incubation mixture. Binding affinities can be determined by observing the level of specific complexes formed in the presence of a nonradioactive competitor DNA. The kinetics of association and dissociation of a specific DNA–protein complex can also be studied in the filter binding assay. However, filter binding assays cannot differentiate between dissimilar DNA–protein complexes, so this assay is effective only when one DNA–protein complex is formed. In addition, the DNA-binding protein must not be denatured during the purification process, because the filter binding assay is dependent on the interaction between the DNA-binding protein and a specific DNA sequence.

 Because various membrane filters differ in binding specificities, the ability of nitrocellulose membranes to bind proteins and not native DNA is critical to the success of the filter binding assay. However, the molecular basis for this discrimination is not well understood. Both electrostatic and hydrophobic interactions have been suggested to be involved in the binding of macromolecules to nitrocellulose, but hydrophobic interactions are assumed to play the dominant role in the binding process. Although native DNA does not bind to nitrocellulose, heat-denatured DNA will (3) and should not be used in this assay. In addition, the native DNA tested in the assay must be free of contaminating proteins that could bind the DNA to the nitrocellulose membrane in the absence of the DNA-binding protein being tested.

 Most proteins bind to nitrocellulose, but individual binding affinities are dependent on the surface characteristics of the particular protein. Therefore, the retention of a particular DNA–protein complex on the nitrocellulose filter will depend on the surface characteristics of the protein, the binding capacity of the nitrocellulose membrane, the time that the DNA–protein complex has to interact with the filter, and the regimen used to wash the filter. A rapid flow rate through the filter may not permit the binding of some DNA–protein complexes to the nitrocellulose membrane, and extensive washing of the filter may remove DNA–protein complexes with low affinities for nitrocellulose. The optimum conditions needed to bind a DNA–protein complex to the nitrocellulose membrane will be different for each protein and should be investigated for each filter binding assay.

References

1. O. W. Jones and P. Berg (1966) J. Mol. Biol. 22, 199–209. 

2. A. D. Riggs, H. Suzuki, and S. Bourgeois (1970) J. Mol. Biol. 48, 67–83. 

3. A. P. Nygaard and B. D. Hall (1963) Biochem. Biophys. Res. Commun. 12, 98–104.