Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes.
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.
Herlt
M
M
Institut für Virologie, Universität zu Köln.
Schwarz
H P
HP
Neumann
R
R
Eggers
H J
HJ
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005374:Filtration / Q000295:instrumentation; D010761:Phosphorus Radioisotopes; D011485:Protein Binding
D015342:DNA Probes; D004268:DNA-Binding Proteins; D010761:Phosphorus Radioisotopes; D004247:DNA