[en] Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (Kd) of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.
1. Fried, M. G., and Crothers, D. M. (1985) Nucleic Acids Res. 9, 6505-6525.
2. Klotz, J. L., Minami, R. N., and Teplitz, R. L. (1979) J. Immunol. Methods 29, 155-165.
3. Pisetsky, D. S., and Peters, D. V. (1981) J. Immunol. Methods 41, 187-200.
4. Sabbatini, A. R., Werner, P. A., Guha, C., Paddock, G. V., and Galbraith, R. M. (1993) Biotechniques 15, 706-713.
5. Ding, H., Descheemaeker, K., Marynen, P., Nelles, L., Carvalho, T., Carmo-Fonseca, M., Collen, D., and Belayew, A. (1996) DNA Cell. Biol. 15, 429-442.
6. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31-40.
7. Descheemaeker, K. A., Wyns, S., Nelles, L., Auwerx, J., Ny, T., and Collen, D. (1992) J. Biol. Chem. 267, 15086-15091.
8. Dahlquist, F. W. (1978) Methods Enzymol. 48, 270-299.
9. Engvall, E. (1980) Methods Enzymol. 70, 419-439.
10. Ludwig, L. B., Klinge, C. M., Peale, F. V., Bambara, R. A., Zain, S., and Hilf, R. (1990) Mol. Endocrinol. 4, 1027-1033.
11. Sha, M., Ferre-D'Amare, A. R., Burley, S. K., and Goss, D. J. (1995) J. Biol. Chem. 270, 19325-19329.
12. Carlsson, B., and Haggblad, J. (1995) Anal. Biochem. 232, 172-179.
13. Guan, C., Li, P., Riggs, P. D., and Inouye, H. (1987) Gene 67, 21-30.
14. Hochuli, E., and Piesecki, S. (1992) Methods Companion Methods Enzymol. 4, 68-72.
15. Kolodziej, P. A., and Young, R. A. (1991) Methods Enzymol. 194, 508-519.