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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5428-5432, June 1993 Biochemistry A double-filter method for nitrocellulose-filter binding: Application to protein-nucleic acid interactions (DNA-protein interactions/equilibrium binding) ISAAC WONG AND TIMOTHY M. LOHMAN Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, 660 South Euclid Avenue, St. Louis, MO 63110 Communicated by Robert L. Baldwin, March 12, 1993 ent, additional control experiments are required, compounding any imprecision in the data-collection process. Therefore, while technical improvements leading to increased precision in data acquisition will not eliminate system-dependent problems, they do greatly improve the quality of data. While studying equilibrium binding ofEscherichia coli Rep protein to short oligodeoxynucleotides (7, 8), we developed another approach for performing nitrocellulose-filter binding based on two modifications to the standard technique. (i) The first of these involves the use of a modified 96-well dot-blot apparatus. (ii) In the second and independent improvement, a DEAE anion-exchange membrane is placed directly beneath the nitrocellulose membrane as a trap for DNA not retained by the nitrocellulose. Combined with direct (3emission technology, these modifications dramatically increase both accuracy and precision while greatly reducing the time required for analysis. ABSTRACT Nitrocellulose-fiter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision. To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane. Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate fiters for each titration point. The entire titration can then be quantitated simultaneously with direct two-dimensional «-emission imaging technology. The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered. This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered. The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered. The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia col Rep protein, a helicase, and its interactions with short oligodeoxynucleotides. MATERIALS AND METHODS Buffers, Proteins, and Oligodeoxynucleotides. Solutions were made with reagent-grade chemicals using distilled H20 that was deionized by using a Milli-Q System (Millipore). Binding buffer is 20 mM Tris HCl, pH 7.5 at 4°C/6 mM NaCl/5 mM 2-mercaptoethanol/1 mM Na3EDTA/10% (vol/ vol) glycerol. E. coli Rep protein was purified to >99% as described (17, 18). Its concentration was determined spectrophotometrically, using an extinction coefficient for the monomer of e280 = 7.68 x 104 M-1cm-1 (17).* T4 polynucleotide kinase was from United States Biochemicals. Oligodeoxynucleotides, dT16 and the 16-bp duplex hairpin (HP; 5' -GACTCGTTACCTGAGT-T4-ACTCAGGTAACGAGTC), were synthesized and purified to >99% purity as described (7, 18, 20). Concentrations of dT16 and HP were determined spectrophotometrically in 10 mM Tris HCl, pH 7.5/1 mM EDTA/150 mM NaCl at 25°C using 8260 = 129,600 and 270,000 M-1 cm-1 per molecule, respectively (7). DNA was 5'-endlabeled by using [y-32P]ATP and T4 polynucleotide kinase as described (7, 8). Nitrocellulose and DEAE Filters. Nitrocellulose and DEAE filters (NA45) were obtained from Schleicher & Schuell in precut 4.5 x 5 inch sheets (1 inch = 2.54 cm). To reduce retention of free single-stranded DNA, nitrocellulose filters were presoaked for 10 min in 0.4 M KOH followed by rinsing in Milli-Q H20 until the pH returned to neutral (21, 22). Filters were then equilibrated in binding buffer at 4°C for a minimum of 1 hr before use. New DEAE filters were washed once in 0.1 M Na3EDTA, pH 8.8, and three times in 1.0 M NaCl with agitation for 10 min each followed by a quick rinse (