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Quantitative Nonisotopic Nitrocellulose Filter Binding Assays: Bacterial Manganese Superoxide Dismutase–Dna Interactions, Joshua Czerwinski, Stephanie Hovan, David Mascotti

David P. Mascotti

Nitrocellulose filter binding assays (NCFBAs) have been used for many years to qualitatively and quantitatively determine protein–nucleic acid affinities. While this technique can be robust thermodynamically and fairly simple to perform, the requirement of radiolabeled nucleic acids (typically 32P) has several major drawbacks. Some disadvantages are the short half-life of 32P, the inherent safety concerns, and the cost of working with radioisotopes. Another drawback is that over time the beta emissions cause fragmentation of the nucleic acids. We have modified standard NCFBAs by developing a quantitative nonisotopic chemiluminescent method using biotin-labeled DNA and a dual-filter format. The biotin tag is …