A fluorescence based non-radioactive electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of the most powerful methods for studying protein-DNA interactions. Typically, 32P-labeled DNA probes containing the sequence bound by the protein of interest are used in EMSA (rEMSA). Although rEMSA is sensitive and practicable, it relies on the handling of hazardous radioisotopes, and does not easily allow quantification. We developed a non-radioactive procedure using fluorescence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific probes (fEMSA) and an automatic DNA sequencer for analysis. Testing different DNA-binding proteins (restriction endonuclease EcoRII, transcription factor NFkappaB and it's subunit p50) the results in fEMSA and rEMSA are similar in regard to quality, reproducibility, and sensitivity. fEMSA allows a semiquantitative screening of large amounts of samples for specific DNA binding activities and is, therefore, a high throughput technology for semiquantitative analysis of DNA-protein interaction.
Ruscher
K
K
Klinik für Neurologie, Humboldt Universität zu Berlin, Schumannstrasse 20-21, D-10098, Berlin, Germany.
Reuter
M
M
Kupper
D
D
Trendelenburg
G
G
Dirnagl
U
U
Meisel
A
A
D002232:Carbocyanines; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D015345:Oligonucleotide Probes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D016328:NF-kappa B; D015345:Oligonucleotide Probes; C085321:cyanine dye 5; D004247:DNA; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific