Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes |
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Authors: | M Herlt H P Schwarz R Neumann H J Eggers |
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Institution: | Institut für Virologie, Universit?t zu K?ln. |
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Abstract: | Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions. |
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